ACPL DNA and polypeptides

ABSTRACT

The invention is directed to purified and isolated novel ACPL polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and the uses of the above.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention is directed to purified and isolated ACPLpolypeptides, the nucleic acids encoding such polypeptides, processesfor production of recombinant forms of such polypeptides, antibodiesgenerated against these polypeptides, fragmented peptides derived fromthese polypeptides, the use of such polypeptides and fragmented peptidesas molecular weight markers, the use of such polypeptides and fragmentedpeptides as controls for peptide fragmentation, and kits comprisingthese reagents. The invention is further directed to the use of ACPLpolypeptides, the nucleic acids encoding such polypeptides, andantibodies generated against these polypeptides in the study of cellsignaling in response to IL-18 stimulation, and inducible proteinexpression systems based on the involvement of ACPL polypeptides in cellsignaling.

[0003] 2. Description of Related Art

[0004] The IL-1 type I receptor (IL-1R) mediates the biological effectsof IL-1. Activities attributed to IL-1α and IL-1β include induction ofinflammatory cytokines and other inflammatory responses includingprostaglandins, metalloproteinases, adhesion molecules, acute phaseproteins, hematopoiesis, fever, bone resorption, and Th2 cell growth anddifferentiation.

[0005] IL-1 has been implicated in chronic inflammatory diseases, suchas rheumatoid arthritis and inflammatory bowel disease. There isincreasing evidence that IL-1 plays a role in osteoporosis. All of theseactivities are initiated by the signaling function of the cytoplasmicportion of the type I IL-1R. IL-1ra inhibits the activities of IL-1 bybinding to the type I IL 1 receptor, thereby blocking access to IL-1αand IL-1β while eliciting no biological response of its own.

[0006] IL-18 is a homolog of IL-1α and IL-1β, and may mediate itsactivities via a receptor homologous to IL-1R, IL1 receptor relatedprotein 1 (IL-1 RrpI)(See Parnet et al., J. Biol. Chem 271:3967, 1996,and Torigoe et al., J. Biol. Chem 272:25737, 1997). IL-18 acts as astimulator of Th1 cell growth and differentiation, and is a potentinducer of interferon production from Th1 cells. IL-18 enhances NK cellkilling activity and has been implicated in septic shock, liverdestruction, and diabetes. Furthermore IL-18 exhibits in vivo antitumoreffects in mice, which are immunologically mediated (Micallef et al.,Cancer Immunol. Immunother. 43:361, 1997).

[0007] The discovery and identification of proteins is at the forefrontof modern molecular biology and biochemistry.

[0008] The identification of the primary structure, or sequence, of asample protein is the culmination of an arduous process ofexperimentation. In order to identify an unknown sample protein, theinvestigator can rely upon comparison of the unknown sample protein toknown peptides using a variety of techniques known to those skilled inthe art. For instance, proteins are routinely analyzed using techniquessuch as electrophoresis, sedimentation, chromatography, and massspectrometry.

[0009] Comparison of an unknown protein sample to polypeptides of knownmolecular weight allows a determination of the apparent molecular weightof the unknown protein sample (T. D. Brock and M. T. Madigan, Biology ofMicroorganisms 76-77 (Prentice Hall, 6d ed. 1991)). Protein molecularweight standards are commercially available to assist in the estimationof molecular weights of unknown protein samples (New England BiolabsInc. Catalog: 130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758).However, the molecular weight standards may not correspond closelyenough in size to the unknown sample protein to allow an accurateestimation of apparent molecular weight.

[0010] The difficulty in estimation of molecular weight is compounded inthe case of proteins that are subjected to fragmentation by chemical orenzymatic means (A. L. Lehninger, Biochemistry 106-108 (Worth Books, 2ded. 1981)). Chemical fragmentation can be achieved by incubation of aprotein with a chemical, such as cyanogen bromide, which leads tocleavage of the peptide bond on the carboxyl side of methionine residues(E. Gross, Methods in Enz. 11:238-255, 1967). Enzymatic fragmentation ofa protein can be achieved by incubation of a protein with a proteasethat cleaves at multiple amino acid residues (D. W. Cleveland et al., J.Biol. Chem. 252:1102-1106, 1977). Enzymatic fragmentation of a proteincan also be achieved by incubation of a protein with a protease, such asAchromobacter protease I (F. Sakiyama and A. Nakata, U.S. Pat. No.5,248,599; T. Masaki et al., Biochim. Biophys. Acta 660:44-50, 1981; T.Masaki et al., Biochim. Biophys. Acta 660:51-55, 1981), which leads tocleavage of the peptide bond on the carboxyl side of lysine residues.The molecular weights of the fragmented peptides can cover a large rangeof molecular weights and the peptides can be numerous. Variations in thedegree of fragmentation can also be accomplished (D. W. Cleveland etal., J. Biol. Chem. 252:1102-1106, 1977).

[0011] The unique nature of the composition of a protein with regard toits specific amino acid constituents results in a unique positioning ofcleavage sites within the protein. Specific fragmentation of a proteinby chemical or enzymatic cleavage results in a unique “peptidefingerprint” (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106,1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980). Consequently,cleavage at specific sites results in reproducible fragmentation of agiven protein into peptides of precise molecular weights. Furthermore,these peptides possess unique charge characteristics that determine theisoelectric pH of the peptide. These unique characteristics can beexploited using a variety of electrophoretic and other techniques (T. D.Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall,6d ed. 1991)).

[0012] When a peptide fingerprint of an unknown protein is obtained,this can be compared to a database of known proteins to assist in theidentification of the unknown protein (W. J. Henzel et al., Proc. Natl.Acad. Sci. USA 90:5011-5015, 1993; B. Thiede et al., Electrophoresis1996, 17:588-599, 1996). A variety of computer software programs areaccessible via the Internet to the skilled artisan for the facilitationof such comparisons, such as MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site:www.mann.embl-heiedelberg.de . . . deSearch/FR_PeptideSearchForm.html),and ProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programsallow the user to specify the cleavage agent and the molecular weightsof the fragmented peptides within a designated tolerance. The programscompare these molecular weights to protein databases to assist in theelucidation of the identity of the sample protein. Accurate informationconcerning the number of fragmented peptides and the precise molecularweight of those peptides is required for accurate identification.Therefore, increasing the accuracy in the determination of the number offragmented peptides and the precise molecular weight of those peptidesshould result in enhanced success in the identification of unknownproteins.

[0013] Fragmentation of proteins is further employed for the productionof fragments for amino acid composition analysis and protein sequencing(P. Matsudiara, J. Biol. Chem. 262:10035-10038, 1987; C. Eckerskom etal., Electrophoresis 1988, 9:830-838, 1988), particularly the productionof fragments from proteins with a “blocked” N-terminus. In addition,fragmentation of proteins can be used in the preparation of peptides formass spectrometry (W. J. Henzel et al., Proc. Natl. Aca Sci. USA90:5011-5015, 1993; B. Thiede et al., Electrophoresis 1996, 17:588-599,1996), for immunization, for affinity selection (R. A. Brown, U.S. Pat.No. 5,151,412), for determination of modification sites (e.g.phosphorylation), for generation of active biological compounds (T. D.Brock and M. T. Madigan, Biology of Microorganisms 300-301 (PrenticeHall, 6d ed. 1991)), and for differentiation of homologous proteins (M.Brown et al., J. Gen. Virol. 50:309-316, 1980).

[0014] In view of the continuing interest in protein research and theelucidation of protein structure and properties, there exists a need inthe art for polypeptides suitable for use in peptide fragmentationstudies and in molecular weight measurements.

SUMMARY OF THE INVENTION

[0015] The invention encompasses isolated nucleic acid moleculescomprising the DNA sequences of SEQ ID NO:1 SEQ ID NO:3, the codingregion of SEQ ID NO:6, and isolated nucleic acid molecules encoding theamino acid sequence of SEQ ID NO:2 and SEQ ID NO:7. The invention alsoencompasses nucleic acid molecules complementary to these sequences. Assuch, the invention includes double-stranded nucleic acid moleculescomprising the DNA sequences of SEQ ID NO:1 and the coding region of SEQID NO:6, and isolated nucleic acid molecules encoding the amino acidsequences of SEQ ID NO:2 and SEQ ID NO:7. Both single-stranded anddouble-stranded RNA and DNA ACPL nucleic acid molecules are encompassedby the invention. These molecules can be used to detect bothsingle-stranded and double-stranded RNA and DNA variants of ACPLencompassed by the invention. A double-stranded DNA probe allows thedetection of nucleic acid molecules equivalent to either strand of thenucleic acid molecule. Isolated nucleic acid molecules that hybridize toa denatured, double-stranded DNA comprising the DNA sequence of SEQ IDNO:1 or SEQ ID NO:6, or an isolated nucleic acid molecule encoding theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:7 under conditions ofmoderate stringency in 50% formamide and 6×SSC, at 42° C. with washingconditions of 60° C., 0.5×SSC, 0.1% SDS are encompassed by theinvention.

[0016] The invention further encompasses isolated nucleic acid moleculesderived by in vitro mutagenesis from SEQ ID NO:1 or SEQ ID NO:6. Invitro mutagenesis would include numerous techniques known in the artincluding, but not limited to, site-directed mutagenesis, randommutagenesis, and in vitro nucleic acid synthesis. The invention alsoencompasses isolated nucleic acid molecules degenerate from SEQ ID NO:1and SEQ ID NO:6, as a result of the genetic code; isolated nucleic acidmolecules that are allelic variants of human ACPL DNA, or a specieshomolog of ACPL DNA. The invention also encompasses recombinant vectorsthat direct the expression of these nucleic acid molecules and hostcells transformed or transfected with these vectors.

[0017] The invention also encompasses isolated polypeptides encoded bythese nucleic acid molecules, including isolated polypeptides having amolecular weight of approximately 70 kD as determined by SDS-PAGE andisolated polypeptides in non-glycosylated form. Isolated polyclonal ormonoclonal antibodies that bind to these polypeptides are encompassed bythe invention. The invention further encompasses methods for theproduction of ACPL polypeptides including culturing a host cell underconditions promoting expression and recovering the polypeptide from theculture medium. Especially, the expression of ACPL polypeptides inbacteria, yeast, plant, and animal cells is encompassed by theinvention.

[0018] In addition, assays utilizing ACPL polypeptides to screen forpotential inhibitors of activity associated with ACPL polypeptidecounter-structure molecules or ACPL binding proteins, and methods ofusing ACPL polypeptides as therapeutic agents for the treatment ofdiseases mediated by ACPL polypeptide counter-structure molecules orbinding proteins are encompassed by the invention. Further, methods ofusing ACPL polypeptides in the design of inhibitors thereof are also anaspect of the invention.

[0019] The invention further encompasses the fragmented peptidesproduced from ACPL polypeptides by chemical or enzymatic treatment. Inaddition, forms of ACPL polypeptide molecular weight markers andfragmented peptides thereof, wherein at least one of the sites necessaryfor fragmentation by chemical or enzymatic means has been mutated, arean aspect of the invention.

[0020] The invention also encompasses a method for the visualization ofACPL polypeptide molecular weight markers and fragmented peptidesthereof using electrophoresis. The invention further includes a methodfor using ACPL polypeptide molecular weight markers and fragmentedpeptides thereof as molecular weight markers that allow the estimationof the molecular weight of a protein or a fragmented protein sample. Theinvention further encompasses methods for using ACPL polypeptides andfragmented peptides thereof as markers, which aid in the determinationof the isoelectric point of a sample protein. The invention alsoencompasses methods for using ACPL polypeptides and fragmented peptidesthereof as controls for establishing the extent of fragmentation of aprotein sample.

[0021] Further encompassed by this invention are kits to aid thedetermination of molecular weights of a sample protein utilizing ACPLpolypeptide molecular weight markers, fragmented peptides thereof, andforms of ACPL polypeptide molecular weight markers, wherein at least oneof the sites necessary for fragmentation by chemical or enzymatic meanshas been mutated.

[0022] Also encompassed by this invention are processes associated withinducible protein expression systems based upon ACPL-dependentinduction. Such systems can include, but are not limited to,ACPL-dependent induction of NFkB-mediated signaling in response to IL-18stimulation and Ap-1-mediated signaling in response to IL-18stimulation. Further encompassed within the present invention areprocesses that are associated with responses to the IL-18 induction ofthe MAP kinase family, kinases JNK and p38.

DETAILED DESCRIPTION OF THE INVENTION

[0023] cDNAs encoding mouse and human ACPL polypeptides have beenisolated and are disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 andSEQ ID NO:6. This discovery of the cDNAs encoding ACPL polypeptidesenables construction of expression vectors comprising nucleic acidsequences encoding ACPL polypeptides and ACPL polypeptide fragments;construction of host cells transfected or transformed with theexpression vectors; construction of biologically active ACPLpolypeptides and ACPL molecular weight markers as isolated and purifiedproteins; and the preparation of antibodies immunoreactive with ACPLpolypeptides.

[0024] ACPL nucleotides and polypeptides were obtained as follows. TheIMAGE clone of EST sequence AA203986, which was derived from mousethymus, was obtained and sequenced. This sequence information was usedto screen a cDNA library from mouse T cells (ELA), and a full-lengthmurine clone of ACPL DNA was isolated and sequenced. The ACPL sequencerepresents sequence from at least three independent isolates over theentire open reading frame. The sequence of the coding region of mouseACPL DNA is given in SEQ ID NO:1. The amino acid sequence encoded by SEQID NO:1 is presented in SEQ ID NO:2. The mouse ACPL encoded by SEQ IDNO:1 includes an extracellular domain of 356 amino acids (residues 1-356from N- to C-terminus of SEQ ID NO:2) that includes a signal peptide of14 amino acids (residues 1-14 of SEQ ID NO:2); a transmembrane region of24 amino acids (residues 357-380 of SEQ ID NO:2) and a cytoplasmicdomain of amino acids (residues 381-614 of SEQ ID NO:2).

[0025] The sequence of this clone shows similarity to the sequence ofmembers of the IL-1 receptor family thereby demonstrating that the mouseACPL DNA (SEQ ID NO:1) encodes a receptor (SEQ ID NO:2) that is a memberof the IL-1 receptor family.

[0026] A clone derived from a human NK cell library, QQ1352, wasidentified as a human homolog of mouse ACPL DNA. Clone QQ1352 representsa partially spliced mRNA clone, and part of this sequence is identicalto the sequence of an exon found within a genomic DNA clone from a BAC,that was deposited in Genbank under accession number B64403.Furthermore, this sequence (nucleotides 179-244 of SEQ ID NO:5)corresponds in position to exon 7 of the human type I IL-1R, andindicates an involvement of ACPL polypeptide in mediating inflamatoryresponses.

[0027] The amino acid sequence encoded by the QQ1352 clone was comparedto the mouse ACPL polypeptide sequence. Bestfit comparisons wereperformed with mouse ACPL polypeptide sequence with translations in 3different frames of the QQ1352 sequence, beginning at nucleotide 522. Itis apparent from these alignments that there are at least twoframeshifts leading to significant homology between the mouse and humansequences in all three frames depending on the position in the sequence,demonstrating the QQ1352 contains a portion of human ACPL.

[0028] To obtain full length human ACPL, the human cDNA clone, QQ1352,was used to to probe clones from PBL, PBT and NK cDNA libraries. Theregion of clone QQ1352 used as a probe was homologous to murine ACPLnucleotides 1196 through 1753. A full-length clone was not obtained fromany of the libraries, so vector-anchored PCR was carried out in each ofthe libraries to obtain the 5′ end of the open reading frame. The fullDNA sequence of human ACPL is disclosed in SEQ ID NO:6. The amino acidsequence encoded by SEQ ID NO:6 is presented in SEQ ID NO:7. The ACPLencoded by SEQ ID NO:6 includes an extracellular domain of 356 aminoacids (residues 1-356 from N- to C-terminus of SEQ ID NO:7) thatincludes a signal peptide of 14 amino acids (residues 1-14 of SEQ IDNO:7); a transmembrane region of 25 amino acids (residues 357-381 of SEQID NO:7) and a cytoplasmic domain of amino acids (residues 382-599 ofSEQ ID NO:7).

[0029] The coexpression of ACPL and IL-1Rrp1 results in a dramaticenhancement of NFkB activity in cells stimulated with IL-18. Incontrast, the expression of ACPL or IL-1Rrp1 alone does not result inIL-18 responsiveness. Therefore, ACPL plays a role in mediating IL-18responses, and can be a component of the IL-18 receptor complex. Inaddition, a receptor for IL-18 can be used as an inhibitor of IL-18induced inflammatory responses. Accordingly, an embodiment of thepresent invention includes the extracellular portion

[0030] Preferred DNA and amino acid embodiments of the present inventioninclude the coding region of SEQ ID NO:1 and SEQ ID NO:6 and the aminoacids encoded by SEQ ID NO:1 and SEQ ID NO:6, shown in SEQ ID NO:2 andSEQ ID NO:7, respectively. Additional preferred embodiments are domainsof the ACPL amino acid sequences and the nucleotide sequences thatencode the domains. For SEQ ID NO:7: the domains include: anextracellular domain of 356 amino acids (residues 1-356 from N- toC-terminus of SEQ ID NO:7) that includes a signal peptide of 14 aminoacids (residues 1-14 of SEQ ID NO:7); a transmembrane region of 25 aminoacids (residues 357-381 of SEQ ID NO:7) and a cytoplasmic domain ofamino acids (residues 382-599 of SEQ ID NO:7). For SEQ ID NO:2, the ACPLamino acid sequence domains of the present invention include: anextracellular domain of 356 amino acids (residues 1-356 from N- toC-terminus of SEQ ID NO:2) that includes a signal peptide of 14 aminoacids (residues 1-14 of SEQ ID NO:2); a transmembrane region of 24 aminoacids (residues 357-380 of SEQ ID NO:2) and a cytoplasmic domain ofamino acids (residues 381-614 of SEQ ID NO:2).

[0031] The discovery of the nucleic acids of the invention enables theconstruction of expression vectors comprising nucleic acid sequencesencoding polypeptides; host cells transfected or transformed with theexpression vectors; and isolated and purified biologically activepolypeptides and fragments thereof. Further, the discovery of thedisclosed nucleic acids, and fragments or oligonucleotides thereof,allows their use as probes to identify nucleic acids encoding proteinshaving homology with IL-1 and to identify nucleic acids encodingproteins having a role in IL-18 mediated responses. Moreover, nucleicacids and oligonucleotides of the present invention find use in mappingDNA on human chromosome 2q and to identify genes associated with certaindiseases, syndromes or other human conditions associated with humanchromosome number 2q. The table below identifies a number of suchdiseases, syndromes, or conditions. Location Gene Name Disease Name 2q11Cyclic nucleotide-gated Achromatopsia-2 channel, alpha-3 2q11Dual-specificity phosphatase 2 2q11 Tibial muscular dystrophy Tibialmuscular dystrophy 2q11-q12 RAN-binding protein 2-like 1 2q11-q13Ectodermal dysplasia-3, Ectodermal dysplasia-3, anhidrotic anhidrotic2q11-q14 Solute carrier family 20, phosphate transporter, member 1(Gibbon ape leukemia virus receptor-1) 2q11-q11.2 Sulfotransferase 1C12q11.2 Barren, Drosophila, homolog of, 1 2q11.2 Solute carrier family 9(sodium/hydrogen exchanger, isoform 2) 2q11.2-q12 Lymphoid nuclearprotein related to AF4 2q12 Interleukin-1 receptor, type I 2q12zeta-chain associated protein Selective T cell defect kinase, 70 kD(syk-related tyrosine kinase) 2q12-q14 Four and a half LIM domains- 2(down-regulated in rhabdo- myosarcoma LIM protein) 2q12-q14Immunoglobulin orphon (transposed element) 1 2q12-q14 Paired boxhomeotic gene-8 hypothyroid, congenital, due to thyroid dysgenesisorhypoplasia 2q12-q21 Diazepam-binding inhibitor 2q12-q22 Interleukin-1receptor, type II 2q12-qter Nucleolin 2q13 BENE protein 2q13 mal, T celldifferentiation protein-like 2q13 Nephronophthisis-1 (juvenile)Nephronophthisis-1 (juvenile) 2q13-q14 Protein C (inactivator ofThrombophilia due to coagulation factors protein C deficiency Va andVIIIa) Purpura fulminans, neonatal 2q13-q21 Engrailed-1 2q14Amphiphysin-like (box- dependent MYC-interacting protein-1) 2q14GLI-Kruppel family member GLI2 (oncogene GLI2) 2q14 Interleukin-1 alpha

[0032] The nucleic acids of the present invention enable the use ofsingle-stranded sense or antisense oligonucleotides from the nucleicacids to inhibit expression of polynucleotide encoded by the ACPL gene.The polypeptides and soluble fragments of the present invention (e.g.extracellular domains of SEQ ID NO:2 and SEQ ID NO:7 and activefragments thereof) can be used to inhibit 1118 mediated responses and tostudy processes associated with inducible protein expression systemsthat are based upon ACPL-dependent induction. Such systems can include,but are not limited to, ACPL-dependent induction of NFkB-mediatedsignaling in response to IL-18 stimulation and Ap-1-mediated signalingin response to IL-18 stimulation. Similarly, such processes includethose that are associated with responses to the IL-18 induction of theMAP kinase family kinases JNK and p38.

[0033] Polypeptides and their fragmented peptides of the presentinvention find further use as molecular weight markers, as controlreagents for peptide fragmentation, and as components of kits comprisingthese reagents. Additionally, the polypeptides and olypeptide fragmentsof the present invention are useful in the generation of antibodies.Antibodies so generated are encompassed by the present invention and areuseful as therapeutics and in processes for purifying polypeptides andpolypeptide fragments of the present invention.

[0034] Nucleic Acids

[0035] In one embodiment, the present invention relates to certainisolated nucleotide sequences that are free from contaminatingendogenous material. A “nucleotide sequence” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct. The nucleic acid molecule has beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd sed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

[0036] Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the cDNA ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, or a suitable fragment thereof,as a probe.

[0037] The DNA molecules of the invention include full length genes aswell as polynucleotides and fragments thereof. The full length gene mayinclude the N-terminal signal peptide. Other embodiments include DNAencoding a soluble form, e.g., encoding the extracellular domain of theprotein, either with or without the signal peptide.

[0038] The nucleic acids of the invention are preferentially derivedfrom human sources, but the invention includes those derived fromnon-human species, as well.

[0039] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA sequence can varyfrom that shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:6 and stillencode a polypeptide having the amino acid sequence of SEQ ID NO:2 andSEQ ID NO:7. Such variant DNA sequences can result from silent mutations(e.g., occurring during PCR amplification), or can be the product ofdeliberate mutagenesis of a native sequence.

[0040] The invention thus provides isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:6; (b) DNAencoding the polypeptides of SEQ ID NO:2 and SEQ ID NO:7; (c) DNAcapable of hybridization to a DNA of (a) or (b) under conditions ofmoderate stringency and which encode polypeptides having ACPL activity;(d) DNA capable of hybridization to a DNA of (a) or (b) under conditionsof high stringency and which encode polypeptides of the invention, and(e) DNA which is degenerate as a result of the genetic code to a DNAdefined in (a), (b), (c), or (d) and which encode polypeptides of theinvention. Of course, polypeptides encoded by such DNA sequences areencompassed by the invention.

[0041] As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

[0042] Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

[0043] In another embodiment, the nucleic acid molecules of theinvention also comprise nucleotide sequences that are at least 80%identical to a native sequence. Also contemplated are embodiments inwhich a nucleic acid molecule comprises a sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to a native sequence.

[0044] The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

[0045] The invention also provides isolated nucleic acids useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding an ACPLpolypeptide, or desired fragment thereof may be subcloned into anexpression vector for production of the polypeptide or fragment. The DNAsequence advantageously is fused to a sequence encoding a suitableleader or signal peptide. Alternatively, the desired fragment may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

[0046] The well-known polymerase chain reaction (PCR) procedure also maybe employed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

[0047] Polypeptides and Fragments Thereof

[0048] The invention encompasses polypeptides and fragments thereof invarious forms, including those that are naturally occurring or producedthrough various techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

[0049] The skilled artisan will recognize that the above-describedboundaries of ACPL polypeptide domains (e.g. the extracellular domain,signal peptide, transmembrane region, and cytoplastmic domain) areapproximate and that the boundaries of the transmembrane region and thesignal peptide (which may be predicted by using computer programsavailable for that purpose) may differ from those described above.

[0050] The polypeptides of the invention may be membrane bound or theymay be secreted and thus soluble. Soluble polypeptides are capable ofbeing secreted from the cells in which they are expressed. In general,soluble polypeptides may be identified (and distinguished fromnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the protein.

[0051] In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced. Additional examples of solublepolypeptides are those lacking not only the cytoplasmic domain andtransmembrane region, but also all or part of the above-described spacerregion.

[0052] In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Soluble polypeptides are generally more suitable forintravenous administration. Further, soluble polypeptides can be usefulfor inhibiting activity associated with ACPL binding proteins.

[0053] The invention also provides ACPL polypeptides and fragments,thereof that retain a desired biological activity (e.g. theextracellular domain). Particular embodiments are directed topolypeptide fragments that retain the ability to bind an ACPL bindingprotein or binding partner. Such a fragment may be a solublepolypeptide, as described above. In another embodiment, the polypeptidesand fragments advantageously include regions that are conserved in theIL-1 receptor family as described above.

[0054] Also provided herein are polypeptide fragments comprising atleast 20, or at least 30, contiguous amino acids of the sequence of SEQID NO:2 or SEQ ID NO:7. Fragments derived from the cytoplasmic domainfind use in studies of signal transduction, and in regulating cellularprocesses associated with transduction of biological signals.Polypeptide fragments also may be employed as immunogens, in generatingantibodies.

[0055] Variants

[0056] Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein.

[0057] Variants may exhibit amino acid sequences that are at least 80%identical. Also contemplated are embodiments in which a polypeptide orfragment comprises an amino acid sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to the preferred polypeptide orfragment thereof. Percent identity may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two protein sequences can be determined by comparingsequence information using the GAP computer program, based on thealgorithm of Needleman and Wunsch (J. Mol. Bio. 48:443, 1970) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The preferred default parameters for the GAP program include:(1) a scoring matrix, blosum62, as described by Henikoff and Henikoff(Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3)a gap length weight of 4; and (4) no penalty for end gaps. Otherprograms used by one skilled in the art of sequence comparison may alsobe used.

[0058] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

[0059] Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

[0060] Other derivatives include covalent or aggregative conjugates ofthe polypeptides with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins are discussed below in connection witholigomers. Further, fusion proteins can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys,which is highly antigenic and provides an epitope reversibly bound by aspecific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG®peptide in the presence of certain divalent metal cations, as describedin U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11hybridoma cell line has been deposited with the American Type CultureCollection under accession no. HB 9259. Monoclonal antibodies that bindthe FLAG® peptide are available from Eastman Kodak Co., ScientificImaging Systems Division, New Haven, Conn.

[0061] Among the variant polypeptides provided herein are variants ofnative polypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds toits binding protein or binding partner with essentially the same bindingaffinity as does the native form. Binding affinity can be measured byconventional procedures, e.g., as described in U.S. Pat. No. 5,512,457and as set forth below.

[0062] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

[0063] A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0064] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide.

[0065] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of polypeptides of theinvention in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Further, a given preparation may includemultiple differentially glycosylated species of the protein. Glycosylgroups can be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

[0066] Correspondingly, similar DNA constructs that encode variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences are encompassedby the invention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Alternatively, theSer or Thr can by replaced with another amino acid, such as Ala. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

[0067] In another example of variants, sequences encoding Cys residuesthat are not essential for biological activity can be altered to causethe Cys residues to be deleted or replaced with other amino acids,preventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation.

[0068] Other variants are prepared by modification of adjacent dibasicamino acid residues, to enhance expression in yeast systems in whichKEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0069] Oligomers

[0070] Encompassed by the invention are oligomers or fusion proteinsthat contain ACPL polypeptides. Such oligomers may be in the form ofcovalently-linked or non-covalently-linked multimers, including dimers,trimers, or higher oligomers. As noted above, preferred polypeptides aresoluble and thus these oligomers may comprise soluble polypeptides. Inone aspect of the invention, the oligomers maintain the binding abilityof the polypeptide components and provide therefor, bivalent, trivalent,etc., binding sites.

[0071] One embodiment of the invention is directed to oligomerscomprising multiple polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the polypeptides. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of the polypeptides attached thereto, asdescribed in more detail below.

Immunoglobulin-Based Oligomers

[0072] As one alternative, an oligomer is prepared using polypeptidesderived from immunoglobulins. Preparation of fusion proteins comprisingcertain heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn etal. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11, 1992).

[0073] One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a polypeptide of theinvention to an Fc polypeptide derived from an antibody. A gene fusionencoding the polypeptide/Fc fusion protein is inserted into anappropriate expression vector. Polypeptide/Fc fusion proteins areexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yielddivalent molecules.

[0074] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides made up of the Fc region of an antibodycomprising any or all of the CH domains of the Fc region. Truncatedforms of such polypeptides containing the hinge region that promotesdimerization are also included. Preferred polypeptides comprise an Fcpolypeptide derived from a human IgG1 antibody.

[0075] One suitable Fc polypeptide, described in PCT application WO93/10151 (hereby incorporated by reference), is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. Another useful Fcpolypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and inBaum et al., (EMBO J. 13:39924001, 1994) incorporated herein byreference. The amino acid sequence of this mutein is identical to thatof the native Fc sequence presented in WO 93/10151, except that aminoacid 19 has been changed from Leu to Ala, amino acid 20 has been changedfrom Leu to Glu, and amino acid 22 has been changed from Gly to Ala. Themutein exhibits reduced affinity for Fc receptors.

[0076] The above-described fusion proteins comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Protein A or Protein G columns.

[0077] In other embodiments, the polypeptides of the invention may besubstituted for the variable portion of an antibody heavy or lightchain. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form an oligomer with as many as fourACPL extracellular regions. Alternatively, fusion proteins can beprepared in which ACPL or a soluble fragment of ACPL, e.g. theextracellular region, and IL-1 Rrp1 or a soluble fragment of IL-1Rrp1,e.g. the extracellular region, are substituted for the variable portionof an antibody heavy or light chain.

[0078] Peptide-Linker Based Oligomers

[0079] Alternatively, the oligomer is a fusion protein comprisingmultiple polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated byreference. A DNA sequence encoding a desired peptide linker may beinserted between, and in the same reading frame as, the DNA sequences ofthe invention, using any suitable conventional technique. For example, achemically synthesized oligonucleotide encoding the linker may beligated between the sequences. In particular embodiments, a fusionprotein comprises from two to four soluble ACPL polypeptides, separatedby peptide linkers. Similarly, as described above, the fusion proteincan include two ACPL polypeptides or fragments and two IL-1Rrp1polypeptides or fragments.

[0080] Leucine-Zippers

[0081] Another method for preparing the oligomers of the inventioninvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759, 1988), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize.

[0082] The zipper domain (also referred to herein as an oligomerizing,or oligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). Two nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The products of the nuclear oncogenesfos andjun comprise zipper domains that preferentially form heterodimer (O'Sheaet al., Science 245:646, 1989, Turner and Tjian, Science 243:1689,1989). The zipper domain is necessary for biological activity (DNAbinding) in these proteins.

[0083] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature 338:547, 1989;Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research andHuman Retroviruses 6:703, 1990). The zipper domains in these fusogenicviral proteins are near the transmembrane region of the proteins; it hasbeen suggested that the zipper domains could contribute to theoligomeric structure of the fusogenic proteins. Oligomerization offusogenic viral proteins is involved in fusion pore formation (Spruce etal, Proc. Natl. Acad Sci U.S.A. 88:3523, 1991). Zipper domains have alsobeen recently reported to play a role in oligomerization of heat-shocktranscription factors (Rabindran et al., Science 259:230, 1993).

[0084] Zipper domains fold as short, parallel coiled coils (O'Shea etal., Science 254:539; 1991). The general architecture of the parallelcoiled coil has been well characterized, with a “knobs-into-holes”packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). Thedimer formed by a zipper domain is stabilized by the heptad repeat,designated (abcdefg)_(n) according to the notation of McLachlan andStewart (J. Mol. Biol. 98:293; 1975), in which residues a and d aregenerally hydrophobic residues, with d being a leucine, which line up onthe same face of a helix. Oppositely-charged residues commonly occur atpositions g and e. Thus, in a parallel coiled coil formed from twohelical zipper domains, the “knobs” formed by the hydrophobic sidechains of the first helix are packed into the “holes” formed between theside chains of the second helix.

[0085] The residues at position d (often leucine) contribute largehydrophobic stabilization energies, and are important for oligomerformation (Krystek: et al., Int. J. Peptide Res. 38:229, 1991). Lovejoyet al. (Science 259:1288, 1993) recently reported the synthesis of atriple-stranded α-helical bundle in which the helices run up-up-down.Their studies confirmed that hydrophobic stabilization energy providesthe main driving force for the formation of coiled coils from helicalmonomers. These studies also indicate that electrostatic interactionscontribute to the stoichiometry and geometry of coiled coils. Furtherdiscussion of the structure of leucine zippers is found in Harbury et al(Science 262:1401, 26 Nov. 1993).

[0086] Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, aswell as the leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. (FEBS Letters 344:191, 1994), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al. (Semin. Immunol. 6:267-278, 1994).Recombinant fusion proteins comprising a soluble polypeptide fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomer that forms is recovered from the culture supernatant.

[0087] Certain leucine zipper moieties preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D (SPD)noted above, as described in Hoppe et al. (FEBS Letters 344:191, 1994)and in U.S. Pat. No. 5,716,805, hereby incorporated by reference intheir entirety. This lung SPD-derived leucine zipper peptide comprisesthe amino acid sequence Pro Asp Val Ala Ser Leu Arg Gin Gln Val Glu AlaLeu Gln Gly Gin Val Gln His Leu Gin Ala Ala Phe Ser GIn Tyr.

[0088] Another example of a leucine zipper that promotes trimerizationis a peptide comprising the amino acid sequence Arg Met Lys Gln Ile GluAsp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly Glu Arg, as described in U.S. Pat. No.5,716,805. In one alternative embodiment, an N-terminal Asp residue isadded; in another, the peptide lacks the N-terminal Arg residue.

[0089] Fragments of the foregoing zipper peptides that retain theproperty of promoting oligomerization may be employed as well. Examplesof such fragments include, but are not limited to, peptides lacking oneor two of the N-terminal or C-terminal residues presented in theforegoing amino acid sequences. Leucine zippers may be derived fromnaturally occurring leucine zipper peptides, e.g., via conservativesubstitution(s) in the native amino acid sequence, wherein the peptide'sability to promote oligomerization is retained.

[0090] Other peptides derived from naturally occurring trimeric proteinsmay be employed in preparing oligomeric ACPL, including trimeric ACPL.Alternatively, synthetic peptides that promote oligomerization may beemployed. In particular embodiments, leucine residues in a leucinezipper moiety are replaced by isoleucine residues. Such peptidescomprising isoleucine may be referred to as isoleucine zippers, but areencompassed by the term “leucine zippers” as employed herein.

[0091] Production of Polypeptides and Fragments Thereof

[0092] Expression, isolation and purification of the polypeptides andfragments of the invention may be accomplished by any suitabletechnique, including but not limited to the following:

[0093] Expression Systems

[0094] The present invention also provides recombinant cloning andexpression vectors containing DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising DNA may be used toprepare the polypeptides or fragments of the invention encoded by theDNA. A method for producing polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that the procedure forpurifying the expressed polypeptides will vary according to such factorsas the type of host cells employed, and whether the polypeptide ismembrane-bound or a soluble form that is secreted from the host cell.

[0095] Any suitable expression system may be employed. The vectorsinclude a DNA encoding a polypeptide or fragment of the invention,operably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence. An origin of replication that confersthe ability to replicate in the desired host cells, and a selection geneby which transformants are identified, are generally incorporated intothe expression vector.

[0096] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

[0097] The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site.

[0098] Suitable host cells for expression of polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0099] Prokaryotic Systems

[0100] Prokaryotes include gram-negative or gram-positive organisms.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus.In a prokaryotic host cell, such as E. coli, a polypeptide may includean N-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

[0101] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a DNAsequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, Wis.,USA).

[0102] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λP_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0103] Yeast Systems

[0104] Alternatively, the polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2 μL yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Ampr gene and origin of replication) into theabove-described yeast vectors.

[0105] The yeast α-factor leader sequence may be employed to directsecretion of the polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

[0106] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

[0107] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenineand 80 mg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0108] Mammalian or Insect Systems

[0109] Mammalian or insect host cell culture systems also may beemployed to express recombinant ACPL polypeptides. Bacculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

[0110] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture,1990, pp. 15-69). Additional protocols using commercially availablereagents, such as Lipofectamine lipid reagent (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487-511, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:42164220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0111] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0112] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534and PCT Application WO 97/25420) and the tripartite leader (TPL) and VAgene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem.257:13475-13491, 1982). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow, Current Opinion in Genetics and Development3:295-300, 1993; Ramesh et al., Nucleic Acids Research 24:2697-2700,1996). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by Mosser et al., Biotechniques 22:150-161, 1997, and p2A5Idescribed by Morris et al., Animal Cell Technology, 1997, pp. 529-534.

[0113] A useful high expression vector, pCAVNOT, has been described byMosley et al., Cell 59:335-348, 1989. Other expression vectors for usein mammalian host cells can be constructed as disclosed by Okayama andBerg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSVN1/N4, described by Cosman et al., Nature 312:768, 1984, has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in WO 91/18982, incorporated byreference herein. In yet another alternative, the vectors can be derivedfrom retroviruses.

[0114] Additional useful expression vectors, pFLAG® and pDC311, can alsobe used. FLAG® technology is centered on the fusion of a low molecularweight (lkD), hydrophilic, FLAG® marker peptide to the N-terminus of arecombinant protein expressed by pFLAG® expression vectors. pDC311 isanother specialized vector used for expressing proteins in CHO cells.pDC311 is characterized by a bicistronic sequence containing the gene ofinterest and a dihydrofolate reductase (DHFR) gene with an internalribosome binding site for DHFR translation, an expression augmentingsequence element (EASE), the human CMV promoter, a tripartite leadersequence, and a polyadenylation site.

[0115] Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type 11interleukin-1 receptor signal peptide described in EP 460,846.

[0116] Isolation and Purification

[0117] The invention also includes methods of isolating and purifyingACPL polypeptides and fragments thereof.

[0118] The “isolated” polypeptides or fragments thereof encompassed bythis invention are ACPL polypeptides or fragments that are not in anenvironment identical to an environment in which it or they can be foundin nature. The “purified” polypeptides or fragments thereof encompassedby this invention are essentially free of association with otherproteins or polypeptides, for example, as a purification product ofrecombinant expression systems such as those described above or as apurified product from a non-recombinant source such as naturallyoccurring cells and/or tissues.

[0119] In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fc moietiesdescribed previously.

[0120] With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

[0121] In general, recombinant ACPL polypeptide or fragment can beisolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps. As tospecific ways to accomplish these steps, the culture medium first can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. In addition, achromatofocusing step can be employed. Alternatively, a hydrophobicinteraction chromatography step can be employed. Suitable matrices canbe phenyl or octyl moieties bound to resins. In addition, affinitychromatography with a matrix which selectively binds the recombinantprotein can be employed. Examples of such resins employed are lectincolumns, dye columns, and metal-chelating columns. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

[0122] It is also possible to utilize an affinity column comprising anACPL polypeptide-binding protein, such as a monoclonal antibodygenerated against an ACPL polypeptide or fragment thereof, toaffinity-purify expressed polypeptides. These purified ACPL polypeptidescan be removed from an affinity column using conventional techniques,e.g., in a high salt elution buffer and then dialyzed into a lower saltbuffer for use or by changing pH or other components depending on theaffinity matrix utilized, or be competitively removed using thenaturally occurring substrate of the affinity moiety, such as apolypeptide derived from the invention.

[0123] In this aspect of the invention, an ACPL polypeptide-bindingproteins, such as the anti-polypeptide antibodies of the invention orother proteins that may interact with ACPL polypeptide, can be bound toa solid phase support such as a column chromatography matrix or asimilar substrate suitable for identifying, separating, or purifyingcells that express polypeptides of the invention on their surface.Adherence of ACPL polypeptide-binding proteins to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having ACPLpolypeptides on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0124] Alternatively, mixtures of cells suspected of containing ACPLpolypeptide-expressing cells first can be incubated with a biotinylatedACPL polypeptide-binding protein. Incubation periods are typically atleast one hour in duration to ensure sufficient binding to polypeptidesof the invention. The resulting mixture then is passed through a columnpacked with avidin-coated beads, whereby the high affinity of biotin foravidin provides the binding of the polypeptide-binding cells to thebeads. Use of avidin-coated beads is known in the art. See Berenson, etal. J. Cell. Biochem., 10D:239 (1986). Wash of unbound material and therelease of the bound cells is performed using conventional methods.

[0125] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0126] Assays

[0127] The purified polypeptides of the invention (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind in any suitable assay, such as aconventional binding assay. To illustrate, the polypeptide may belabeled with a detectable reagent (e.g., a radionuclide, chromophore,enzyme that catalyzes a colorimetric or fluorometric reaction, and thelike). The labeled polypeptide is contacted with cells expressing asuitable ACPL binding protein, e.g. an anti-ACPL antibody. The cellsthen are washed to remove unbound labeled polypeptide, and the presenceof cell-bound label is determined by a suitable technique, chosenaccording to the nature of the label.

[0128] One example of a binding assay procedure is as follows. Arecombinant expression vector containing an ACPL binding protein cDNA isconstructed. CV1-EBNA-1 cells in 10 cm² dishes are transfected with therecombinant expression vector. CV-1/EBNA-1 cells (ATCC CRL 10478)constitutively express EBV nuclear antigen-1 driven from the CMVimmediate-early enhancer/promoter. CV1-EBNA-1 was derived from theAfrican Green Monkey kidney cell line CV-1 (ATCC CCL 70), as describedby McMahan et al. (EMBO J. 10:2821, 1991).

[0129] The transfected cells are cultured for 24 hours, and the cells ineach dish then are split into a 24-well plate. After culturing anadditional 48 hours, the transfected cells (about 4×10⁴ cells/well) arewashed with BM-NFDM, which is binding medium (RPMI 1640 containing 25mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) towhich 50 mg/ml nonfat dry milk has been added. The cells then areincubated for 1 hour at 37° C. with various concentrations of, forexample, a soluble polypeptide/Fc fusion protein made as set forthabove. Cells then are washed and incubated with a constant saturatingconcentration of a ¹²⁵I-mouse anti-human IgG in binding medium, withgentle agitation for 1 hour at 37° C. After extensive washing, cells arereleased via trypsinization.

[0130] The mouse anti-human IgG employed above is directed against theFc region of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any polypeptide/Fc protein that has bound to the cells. Inall assays, non-specific binding of ¹²⁵-antibody is assayed in theabsence of the Fc fusion protein/Fc, as well as in the presence of theFc fusion protein and a 200-fold molar excess of unlabeled mouseanti-human IgG antibody.

[0131] Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogammacounter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660,1949) are generated on RS/1 (BBN Software, Boston, Mass.) run on aMicrovax computer.

[0132] Another type of suitable binding assay is a competitive bindingassay. To illustrate, biological activity of an ACPL variant may bedetermined by assaying for the variant's ability to compete with thenative protein for binding to an ACPL binding protein.

[0133] Competitive binding assays can be performed by conventionalmethodology. Reagents that may be employed in competitive binding assaysinclude radiolabeled ACPL and intact cells expressing an ACPL bindingprotein (endogenous or recombinant) on the cell surface. For example, aradiolabeled soluble ACPL fragment can be used to compete with a solubleACPL variant for binding to cell surface ACPL binding protein. Insteadof intact cells, one could substitute a soluble ACPL binding protein/Fcfusion protein bound to a solid phase through the interaction of ProteinA or Protein G (on the solid phase) with the Fc moiety. Chromatographycolumns that contain Protein A and Protein G include those availablefrom Pharmacia Biotech, Inc., Piscataway, N.J.

[0134] Another type of competitive binding assay utilizes radiolabeledsoluble ACPL binding protein, such as a ACPL binding protein/Fc fusionprotein, and intact cells expressing ACPL. Qualitative results can beobtained by competitive autoradiographic plate binding assays, whileScatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) may beutilized to generate quantitative results.

[0135] Use of ACPL Nucleic Acid or Oligonucleotides

[0136] In addition to being used to express polypeptides as describedabove, the nucleic acids of the invention, including DNA, andoligonucleotides thereof can be used:

[0137] to identify human chromosome number 2q

[0138] to map genes on human chromosome number 2q

[0139] to identify genes associated with certain diseases, syndromes, orother conditions associated with human chromosome number 2q;

[0140] as single-stranded sense or antisense oligonucleotides, toinhibit expression of polypeptide encoded by the ACPL gene;

[0141] to help detect defective genes in an individual; and

[0142] for gene therapy.

[0143] Probes

[0144] Among the uses of nucleic acids of the invention is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60,contiguous nucleotides of a DNA sequence.

[0145] Because homologs of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4 andSEQ ID NO:6 from other mammalian species are contemplated herein, probesbased on the DNA sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 andSEQ ID NO:6 may be used to screen cDNA libraries derived from othermammalian species, using conventional cross-species hybridizationtechniques.

[0146] Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

[0147] Chromosome Mapping

[0148] All or a portion of the nucleic acids of SEQ ID NO:1, SEQ IDNO:3, and SEQ ID NO:6, including oligonucleotides, can be used by thoseskilled in the art using well-known techniques to identify the human 2q,and the specific locus thereof, that contains the DNA of IL-1R familymembers, including IL-1 receptors I and II, ST2 and IL-1Rrp1. Usefultechniques include, but are not limited to, using the sequence orportions, including oligonucleotides, as a probe in various well-knowntechniques such as radiation hybrid mapping (high resolution), in situhybridization to chromosome spreads (moderate resolution), and Southernblot hybridization to hybrid cell lines containing individual humanchromosomes (low resolution).

[0149] For example, chromosomes can be mapped by radiationhybridization. First, PCR is performed using the Whitehead Institute/MITCenter for Genome Research Genebridge4 panel of 93 radiation hybrids(http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/rhmap/genebridge4.html).Primers are used which lie within a putative exon of the gene ofinterest and which amplify a product from human genomic DNA, but do notamplify hamster genomic DNA. The results of the PCRs are converted intoa data vector that is submitted to the Whitehead/MIT Radiation Mappingsite on the internet (http://www-seq.wi.mit.edu). The data is scored andthe chromosomal assignment and placement relative to known Sequence TagSite (STS) markers on the radiation hybrid map is provided. Thefollowing web site provides additional information about radiationhybrid mapping:http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/07-97.1NTRO.html).

[0150] Identifying Associated Diseases

[0151] As set forth below, the DNA of SEQ ID NO:6 has been mapped to thechromosome 2q. That region is associated with specific diseases whichinclude but are not limited to those identified in Table I, above. Thus,the nucleic acid of SEQ ID NO:6 or a fragment thereof can be used by oneskilled in the art using well-known techniques to analyze abnormalitiesassociated with gene mapping to chromosome 2q. This enables one todistinguish conditions in which this marker is rearranged or deleted. Inaddition, nucleotides of SEQ ID NO:6 or a fragment thereof can be usedas a positional marker to map other genes of unknown location.

[0152] The DNA may be used in developing treatments for any disordermediated (directly or indirectly) by defective, or insufficient amountsof, the genes corresponding to the nucleic acids of the invention.Disclosure herein of native nucleotide sequences permits the detectionof defective genes, and the replacement thereof with normal genes.Defective genes may be detected in in vitro diagnostic assays, and bycomparison of a native nucleotide sequence disclosed herein with that ofa gene derived from a person suspected of harboring a defect in thisgene.

[0153] Sense-Anti Sense

[0154] Other useful fragments of the nucleic acids include antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target mRNA (sense)or DNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the DNA ofSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:6. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 toabout 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0155] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that block orinhibit protein expression by one of several means, including enhanceddegradation of the mRNA by RNAseH, inhibition of splicing, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression ofproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0156] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10448, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0157] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, lipofection, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus.

[0158] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0159] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0160] Use of ACPL Polypeptides and Fragmented Polypeptides

[0161] Uses include, but are not limited to, the following:

[0162] Purifying proteins and measuring activity thereof

[0163] Delivery Agents

[0164] Therapeutic and Research Reagents

[0165] Molecular weight and Isoelectric focusing markers

[0166] Controls for peptide fragmentation

[0167] Identification of unknown proteins

[0168] Preparation of Antibodies

[0169] Purification Reagents

[0170] The polypeptides of the invention find use as a proteinpurification reagent. For example, ACPL polypeptides may be attached toa solid support material and used to purify ACPL binding proteins byaffinity chromatography. In particular embodiments, a polypeptide (inany form described herein that is capable of binding to an ACPL bindingprotein) is attached to a solid support by conventional procedures. Asone example, chromatography columns containing functional groups thatwill react with functional groups on amino acid side chains of proteinsare available (Pharmacia Biotech, Inc., Piscataway, N.J.). In analternative, a polypeptide/Fc protein (as discussed above) is attachedto Protein A- or Protein G-containing chromatography columns throughinteraction with the Fc moiety.

[0171] The polypeptide also finds use in purifying or identifying cellsthat express an ACPL binding protein on the cell surface. Polypeptidesare bound to a solid phase such as a column chromatography matrix or asimilar suitable substrate. For example, magnetic microspheres can becoated with the polypeptides and held in an incubation vessel through amagnetic field. Suspensions of cell mixtures containing ACPL bindingprotein expressing cells are contacted with the solid phase having thepolypeptides thereon. Cells expressing ACPL binding protein on the cellsurface bind to the fixed polypeptides, and unbound cells then arewashed away.

[0172] Alternatively, the polypeptides can be conjugated to a detectablemoiety, then incubated with cells to be tested for binding proteinexpression. After incubation, unbound labeled matter is removed and thepresence or absence of the detectable moiety on the cells is determined.

[0173] In a further alternative, mixtures of cells suspected ofcontaining ACPL binding protein cells are incubated with biotinylatedpolypeptides. Incubation periods are typically at least one hour induration to ensure sufficient binding. The resulting mixture then ispassed through a column packed with avidin-coated beads, whereby thehigh affinity of biotin for avidin provides binding of the desired cellsto the beads. Procedures for using avidin-coated beads are known (seeBerenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing to removeunbound material, and the release of the bound cells, are performedusing conventional methods.

[0174] Measuring Activity

[0175] Polypeptides also find use in measuring the biological activityof ACPL binding protein in terms of their binding affinity. Thepolypeptides thus may be employed by those conducting “qualityassurance” studies, e.g., to monitor shelf life and stability of proteinunder different conditions. For example, the polypeptides may beemployed in a binding affinity study to measure the biological activityof an ACPL binding protein that has been stored at differenttemperatures, or produced in different cell types. The proteins also maybe used to determine whether biological activity is retained aftermodification of an ACPL binding protein (e.g., chemical modification,truncation, mutation, etc.). The binding affinity of the modified ACPLbinding protein is compared to that of an unmodified ACPL bindingprotein to detect any adverse impact of the modifications on biologicalactivity of ACPL binding protein. The biological activity of an ACPLbinding protein thus can be ascertained before it is used in a researchstudy, for example.

[0176] Delivery Agents

[0177] The polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing an ACPL binding protein. Thepolypeptides thus can be used to deliver diagnostic or therapeuticagents to such cells (or to other cell types found to express ACPLbinding protein on the cell surface) in in vitro or in vivo procedures.

[0178] Detectable (diagnostic) and therapeutic agents that may beattached to a polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a colorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins suchas trichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re,¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

[0179] Such agents may be attached to the polypeptide by any suitableconventional procedure. The polypeptide comprises functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to polypeptides by using a suitablebifunctional chelating agent, for example.

[0180] Conjugates comprising polypeptides and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

[0181] Therapeutic Agents

[0182] Polypeptides of the invention may be used in developingtreatments for any disorder mediated (directly or indirectly) bydefective, excessive or insufficient amounts of ACPL or any bindingpartners, including IL polypeptides. Isolated and purified ACPLpolypeptides or a fragment thereof can also be useful itself as atherapeutic agent in inhibiting IL-1 and TNF signaling. Such therapeuticuses of ACPL can involve their administration by the instruction of theACPL polypeptide or fragment into the intracellular environment bywell-known means. One such mean is by encasing the protein in liposomesor coupling it to a monoclonal antibody targeted to a specific celltype.

[0183] The polypeptides may also be employed in inhibiting a biologicalactivity of an ACPL binding protein, in in vitro or in vivo procedures.For example, an ACPL purified polypeptide or soluble fragment thereofmay be used to inhibit binding of an ACPL binding protein to endogenouscell surface ACPL binding partner. Biological effects that result fromthe binding of binding partner to endogenous receptors thus areinhibited.

[0184] In addition, ACPL polypeptides may be administered to a mammal totreat a disorder mediated by an ACPL binding partner. Such bindingpartner-mediated disorders include conditions caused (directly orindirectly) or exacerbated by the binding partner.

[0185] Compositions of the present invention may contain a polypeptidein any form described herein, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble polypeptide or anoligomer comprising soluble ACPL polypeptides, e.g. the extracellulardomain of ACPL or biologically active fragments thereof that bind to thebinding partner.

[0186] Compositions comprising an effective amount of an ACPLpolypeptide or fragment thereof of the present invention, in combinationwith other components such as a physiologically acceptable diluent,carrier, or excipient, are provided herein. The polypeptides can beformulated according to known methods used to prepare pharmaceuticallyuseful compositions. They can be combined in admixture, either as thesole active material or with other known active materials suitable for agiven indication, with pharmaceutically acceptable diluents (e.g.,saline, Tris-HCl, acetate, and phosphate buffered solutions),preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers,solubilizers, adjuvants and/or carriers. Suitable formulations forpharmaceutical compositions include those described in Remington'sPharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton,Pa.

[0187] In addition, such compositions can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0188] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, or by inhalation. Theterm “parenteral” includes injection, e.g., by subcutaneous,intravenous, intracellularly or intramuscular routes, also includinglocalized administration, e.g., at a site of disease or injury.Sustained release from implants is also contemplated. One skilled in thepertinent art will recognize that suitable dosages will vary, dependingupon such factors as the nature of the disorder to be treated, thepatient's body weight, age, and general condition, and the route ofadministration. Preliminary doses can be determined according to animaltests, and the scaling of dosages for human administration is performedaccording to art-accepted practices.

[0189] Compositions comprising nucleic acids in physiologicallyacceptable formulations are also contemplated. DNA may be formulated forinjection, for example.

[0190] Research Agents

[0191] Another use of the polypeptide of the present invention is as aresearch tool for studying the biological effects that result frominhibiting ACPL binding partner/ACPL interactions on different celltypes. Polypeptides also may be employed in in vitro assays fordetecting ACPL binding partner or ACPL or the interactions thereof.

[0192] ACPL polypeptides, and antibodies against ACPL polypeptides canbe used as reagents in a variety of research protocols. A sample of suchresearch protocols are given in Sambrook et al. Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press,(1989). For example, these reagents can serve as markers for cellspecific or tissue specific expression of RNA or proteins. Similarly,these reagents can be used to investigate constituitive and transientexpression of ACPL RNA or polypeptides. ACPL DNA can be used todetermine the chromosomal location of ACPL DNA and to map genes inrelation to this chromosomal location. ACPL DNA can also be used toexamine genetic heterogeneity and heredity through the use of techniquessuch as genetic fingerprinting, as well as to identify risks associatedwith genetic disorders. ACPL DNA can be further used to identifyadditional genes related to ACPL DNA and to establish evolutionary treesbased on the comparison of sequences. ACPL DNA and polypeptides can beused to select for those genes or proteins that are homologous to ACPLDNA or polypeptides, through positive screening procedures such asSouthern blotting and immunoblotting and through negative screeningprocedures such as subtraction.

[0193] ACPL polypeptides can also be used as a reagent to identify (a)any protein that ACPL polypeptide regulates, and (b) other proteins withwhich it might interact. ACPL polypeptides could be used by couplingrecombinant protein to an affinity matrix, or by using them as a bait inthe 2-hybrid system. ACPL polypeptides and fragments thereof can be usedas reagents in the study of signaling pathways used by receptors of theIL-1R family and to block signaling by IL-18 and possibly other ligandsof the IL-1 family.

[0194] Another embodiment of the invention relates to uses of ACPL tostudy cell signal transduction. ACPL polypeptides play a role in immuneresponses which includes cellular signal transduction. As such,alterations in the expression and/or activation of ACPL can haveprofound effects on a plethora of cellular processes. Expression ofcloned ACPL, functionally inactive mutants of ACPL can be used toidentify the role a particular protein plays in mediating specificsignaling events.

[0195] Cellular signaling often involves a molecular activation cascade,during which a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates. These substrates can themselves be kinases which becomeactivated following phosphorylation. Alternatively, they can be adaptormolecules that facilitate down stream signaling through protein-proteininteraction following phosphorylation. Regardless of the nature of thesubstrate molecule(s), expressed functionally active versions of ACPLcan be used in assays such as the yeast 2-hybrid assay to identify whatsubstrate(s) were recognized and activated by ACPL binding partners. Assuch, these novel ACPL can be used as reagents to identify novelmolecules involved in signal transduction pathways.

[0196] Furthermore, ACPL polypeptides and fragments thereof can be usedas reagents in the study of the IL-18 signaling pathway as a reagent toblock IL-18 signaling. The discovery that ACPL polypeptide plays a rolein NFkB signaling allows the use of ACPL polypeptides in studies of NFkBsignaling, particularly with regard to the induction of NFkB signalingby IL-18. The discovery of ACPL polypeptide and its role in NFKBsignaling further allows its use as a reagent in research protocols toelucidate the role of IL-1Rrp1 and IL-18 in cell signaling.

[0197] IL-18 induces many additional signaling responses. Among suchsignaling responses is the induction of the MAP kinase family kinasesJNK and p38. Thus, ACPL polypeptides and fragments thereof can be usedas reagents in the study IL-18 induced signaling responses of the MAPkinase family kinases JNK and p38.

[0198] The discovery that ACPL polypeptide stimulates production of aspecific protein in response to IL-18 allows the generation of inducibleprotein expression systems. In one embodiment, the gene encoding theprotein of interest can be placed within a vector containing three NFkBsites mediating expression of the protein. The skilled artisanrecognizes that many different vectors can be used to achieve expressionlinked to NFkB expression, depending upon the cell type desired forexpression. As an example, 10⁷ S49.1 cells can be transfected byelectroporation in 0.7 ml with 40 μg of a NFkB-linked expression vectorand 20 μg of expression vectors encoding murine ACPL polypeptide andmurine IL-1R-Rp1 at 960 μF and 320V. Cells can be incubated for 2 daysand can then be stimulated with 40 ng/ml murine IL-18 (PeproTech). Theaddition of IL-18 can induce expression of the NFkB-linked gene300-fold. It is understood that many different approaches can be used toacheive induction of protein expression using ACPL polypeptide and IL-18stimulation, and that this embodiment in no way limits the scope of theinvention.

[0199] Due to the regulated production of the NFkB-linked protein, thelevel of this protein within the cells can be modulated according tostimulation with IL-18. The use of a marker gene, such as luciferase,allows the elucidation of inhibitors and regulators of IL-18 stimulatedNFkB signaling. Additionally, control of the level and timing of proteinexpression enables one skilled in the art to examine both the temporaland cumulative effects of the protein of interest. Antibodies againstACPL polypeptides can further be used to inhibit IL-18 stimulated NFkBsignaling in these experiments, allowing a more detailed analysis of thesteps involved in NFkB signaling.

[0200] The purified ACPL polypeptides according to the invention willfacilitate the discovery of inhibitors of ACPL polypeptides. The use ofa purified ACPL polypeptide in the screening of potential inhibitorsthereof is important and can eliminate or reduce the possibility ofinterfering reactions with contaminants.

[0201] In addition, ACPL polypeptides can be used for structure-baseddesign of ACPL polypeptide-inhibitors. Such structure-based design isalso known as “rational drug design.” The ACPL polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of ACPL polypeptide structural informationin molecular modeling software systems to assist in inhibitor design andinhibitor-ACPL polypeptide interaction is also encompassed by theinvention. Such computer-assisted modeling and drug design can utilizeinformation such as chemical conformational analysis, electrostaticpotential of the molecules, protein folding, etc. For example, most ofthe design of class-specific inhibitors of metalloproteases has focusedon attempts to chelate or bind the catalytic zinc atom. Syntheticinhibitors are usually designed to contain a negatively-charged moietyto which is attached a series of other groups designed to fit thespecificity pockets of the particular protease. A particular method ofthe invention comprises analyzing the three dimensional structure ofACPL polypeptides for likely binding sites of substrates, synthesizing anew molecule that incorporates a predictive reactive site, and assayingthe new molecule as described above.

[0202] Molecular Weight, Isoelectric Point Markers

[0203] The present invention further includes processes that utilizeACPL polypeptides as molecular weight markers to estimate the apparentmolecular weight of a sample protein by gel electrophoresis. An isolatedand purified mouse ACPL polypeptide molecular weight marker according tothe invention has a molecular weight of approximately 70,048 Daltons inthe absence of glycosylation. The ACPL polypeptide, together with asample protein, can be resolved by denaturing polyacrylamide gelelectrophoresis by conventional means (U. K. Laemmli, Nature227:680-685, 1970) in two separate lanes of a gel containing sodiumdodecyl sulfate and a concentration of acrylamide between 6-20%.Proteins on the gel can be visualized using a conventional stainingprocedure. The ACPL polypeptide molecular weight marker can be used as amolecular weight marker in the estimation of the apparent molecularweight of the sample protein. The unique amino acid sequence of mouseACPL (SEQ ID NO:2) specifies a molecular weight of approximately 70,048Daltons. Therefore, the ACPL polypeptide molecular weight marker servesparticularly well as a molecular weight marker for the estimation of theapparent molecular weight of sample proteins that have apparentmolecular weights close to 70,048 Daltons. The use of this polypeptidemolecular weight marker allows an increased accuracy in thedetermination of apparent molecular weight of proteins that haveapparent molecular weights close to 70,048 Daltons. Similarly, humanACPL polypeptide (SEQ ID NO:7) can be used as a molecular weight markerfor the estimation of the apparent molecular weight of sample proteins.It is understood of course that many different techniques can be usedfor the determination of the molecular weight of a sample protein usingACPL polypeptides and that this embodiment in no way limits the scope ofthe invention.

[0204] Another preferred embodiment of the invention is the use of ACPLfragmented peptide molecular weight markers, generated by chemicalfragmentation of ACPL polypeptide, as molecular weight markers toestimate the apparent molecular weight of a sample protein by gelelectrophoresis. Isolated and purified ACPL polypeptide can be treatedwith cyanogen bromide under conventional conditions that result infragmentation of the ACPL polypeptide molecular weight marker byspecific hydrolysis on the carboxyl side of the methionine residueswithin the ACPL polypeptide (E. Gross, Methods in Enz. 11:238-255,1967). Due to the unique amino acid sequence of the ACPL polypeptide,the fragmentation of ACPL polypeptide molecular weight markers withcyanogen bromide generates a unique set of ACPL fragmented peptidemolecular weight markers. For instance, human and mouse ACPLpolypeptides will each generate a unique set of ACPL fragmented peptidemolecular weight markers. The sizes of these molecular weight markerscan be predicted using available computer programs. The distribution ofmethionine residues determines the number of amino acids in each peptideand the unique amino acid composition of each peptide determines itsmolecular weight.

[0205] The unique set of ACPL fragmented peptide molecular weightmarkers generated by treatment of mouse ACPL polypeptide with cyanogenbromide comprises 10 fragmented peptides of at least 10 amino acids insize. The peptide encoded by amino acids 2-87 of SEQ ID NO:2 has amolecular weight of approximately 9,724 Daltons. The peptide encoded byamino acids 88-105 of SEQ ID NO:2 has a molecular weight ofapproximately 2,020 Daltons. The peptide encoded by amino acids 111-136of SEQ ID NO:2 has a molecular weight of approximately 3,094 Daltons.The peptide encoded by amino acids 137-188 of SEQ ID NO:2 has amolecular weight of approximately 5,502 Daltons. The peptide encoded byamino acids 189-207 of SEQ ID NO:2 has a molecular weight ofapproximately 2,354 Daltons. The peptide encoded by amino acids 208-299of SEQ ID NO:2 has a molecular weight of approximately 10,617 Daltons.The peptide encoded by amino acids 300-369 of SEQ ID NO:2 has amolecular weight of approximately 8,293 Daltons. The peptide encoded byamino acids 370-558 of SEQ ID NO:2 has a molecular weight ofapproximately 21,559 Daltons. The peptide encoded by amino acids 568-593of SEQ ID NO:2 has a molecular weight of approximately 2,963 Daltons.The peptide encoded by amino acids 594-614 of SEQ ID NO:2 has amolecular weight of approximately 2,343 Daltons.

[0206] Therefore, cleavage of the mouse ACPL polypeptide by chemicaltreatment with cyanogen bromide generates a unique set of ACPLfragmented peptide molecular weight markers. The unique and known aminoacid sequence of these ACPL fragmented peptides allows the determinationof the molecular weight of these fragmented peptide molecular weightmarkers. In this particular case, ACPL fragmented peptide molecularweight markers have molecular weights of approximately 9,724; 2,020;3,094; 5,502; 2,354; 10,617; 8,293; 21,559; 2,963; and 2,343 Daltons.

[0207] The ACPL fragmented peptide molecular weight markers, togetherwith a sample protein, can be resolved by denaturing polyacrylamide gelelectrophoresis by conventional means in two separate lanes of a gelcontaining sodium dodecyl sulfate and a concentration of acrylamidebetween 10-20%. Proteins on the gel can be visualized using aconventional staining procedure. The ACPL fragmented peptide molecularweight markers can be used as molecular weight markers in the estimationof the apparent molecular weight of the sample protein. The unique aminoacid sequence of mouse ACPL specifies a molecular weight ofapproximately 9,724; 2,020; 3,094; 5,502; 2,354; 10,617; 8,293; 21,559;2,963; and 2,343 Daltons for the ACPL fragmented peptide molecularweight markers. Therefore, the ACPL fragmented peptide molecular weightmarkers serve particularly well as molecular weight markers for theestimation of the apparent molecular weight of sample proteins that haveapparent molecular weights close to 9,724; 2,020; 3,094; 5,502; 2,354;10,617; 8,293; 21,559; 2,963; or 2,343 Daltons. Consequently, the use ofthese fragmented peptide molecular weight markers allows an increasedaccuracy in the determination of apparent molecular weight of proteinsthat have apparent molecular weights close to 9,724; 2,020; 3,094;5,502; 2,354; 10,617; 8,293; 21,559; 2,963; or 2,343 Daltons. It isunderstood of course that the unique amino acid sequence of human ACPLpolypeptide (SEQ ID NO:13) can similarly be used to generate a uniqueset of human ACPL fragmented peptide molecular weight markers. Thefragment sizes can readily be determined using available computerprograms.

[0208] In a further embodiment, the sample protein and the ACPLpolypeptide can be simultaneously, but separately, treated with cyanogenbromide under conventional conditions that result in fragmentation ofthe sample protein and the ACPL polypeptide by specific hydrolysis onthe carboxyl side of the methionine residues within the sample proteinand the ACPL polypeptide. As described above, the ACPL fragmentedpeptide molecular weight markers generated by cleavage of the ACPLpolypeptide with cyanogen bromide have molecular weights ofapproximately 9,724; 2,020; 3,094; 5,502; 2,354; 10,617; 8,293; 21,559;2,963; and 2,343 Daltons.

[0209] The fragmented peptides from both the ACPL polypeptide and thesample protein can be resolved by denaturing polyacrylamide gelelectrophoresis by conventional means in two separate lanes of a gelcontaining sodium dodecyl sulfate and a concentration of acrylamidebetween 10-20%. Fragmented peptides on the gel can be visualized using aconventional staining procedure. The ACPL fragmented peptide molecularweight markers can be used as molecular weight markers in the estimationof the apparent molecular weight of the fragmented proteins derived fromthe sample protein. As discussed above, the ACPL fragmented peptidemolecular weight markers serve particularly well as molecular weightmarkers for the estimation of the apparent molecular weight offragmented peptides that have apparent molecular weights close to 9,724;2,020; 3,094; 5,502; 2,354; 10,617; 8,293; 21,559; 2,963; or 2,343Daltons. Consequently, the use of these ACPL fragmented peptidemolecular weight markers allows an increased accuracy in thedetermination of apparent molecular weight of fragmented peptides thathave apparent molecular weights close to 9,724; 2,020; 3,094; 5,502;2,354; 10,617; 8,293; 21,559; 2,963; or 2,343 Daltons. The extent offragmentation of the ACPL polypeptide is further used as a control todetermine the conditions expected for complete fragmentation of thesample protein. It is understood of course that many chemicals could beused to fragment ACPL polypeptides and that this embodiment in no waylimits the scope of the invention.

[0210] In another embodiment, unique sets of ACPL fragmented peptidemolecular weight markers can be generated from ACPL polypeptide usingenzymes that cleave the polypeptide at specific amino acid residues. Dueto the unique nature of the amino acid sequence of the ACPL polypeptide,cleavage at different amino acid residues will result in the generationof different sets of fragmented peptide molecular weight markers.

[0211] An isolated and purified ACPL polypeptide can be treated withAchromobacter protease I under conventional conditions that result infragmentation of the ACPL polypeptide by specific hydrolysis on thecarboxyl side of the lysine residues within the ACPL polypeptide (T.Masaki et al., Biochim. Biophys. Acta 660:44-50, 1981; T. Masaki et al.,Biochim. Biophys. Acta 660:51-55, 1981). Due to the unique amino acidsequence of the ACPL polypeptide, the fragmentation of ACPL polypeptidemolecular weight markers with Achromobacter protease I generates aunique set of ACPL fragmented peptide molecular weight markers. Thedistribution of lysine residues determines the number of amino acids ineach peptide and the unique amino acid composition of each peptidedetermines its molecular weight.

[0212] The unique set of ACPL fragmented peptide molecular weightmarkers generated by treatment of mouse ACPL polypeptide withAchromobacter protease I comprises 20 fragmented peptides of at least 10amino acids in size. The generation of 20 fragmented peptides with thisenzyme treatment of the ACPL polypeptide, as compared to the generationof 10 fragmented peptides with cyanogen bromide treatment of the ACPLpolypeptide, clearly illustrate that the sizes of the fragmented peptidemolecular weight markers will vary depending upon the fragmentationtreatment utilized to fragment the ACPL polypeptide. Both the size andnumber of these fragments are dictated by the amino acid sequence of theACPL polypeptide. Consequently, the number of fragmented peptides willalso vary depending upon the fragmentation treatment utilized tofragment the ACPL polypeptide. In addition, fragmentation of human ACPLpolypeptide (SEQ ID NO:7) will result in unique sets of fragmentedpeptides, dictated by the amino acid sequence of the ACPL polypeptide.

[0213] The peptide encoded by amino acids 1-16 of SEQ ID NO:2 has amolecular weight of approximately 1,897 Daltons. The peptide encoded byamino acids 17-28 of SEQ ID NO:2 has a molecular weight of approximately1,236 Daltons. The peptide encoded by amino acids 30-55 of SEQ ID NO:2has a molecular weight of approximately 3,100 Daltons. The peptideencoded by amino acids 56-71 of SEQ ID NO:2 has a molecular weight ofapproximately 1,721 Daltons. The peptide encoded by amino acids 79-141of SEQ ID NO:2 has a molecular weight of approximately 7,285 Daltons.The peptide encoded by amino acids 148-192 of SEQ ID NO:2 has amolecular weight of approximately 4,893 Daltons. The peptide encoded byamino acids 203-238 of SEQ ID NO:2 has a molecular weight ofapproximately 4,123 Daltons. The peptide encoded by amino acids 250-266of SEQ ID NO:2 has a molecular weight of approximately 1,866 Daltons.The peptide encoded by amino acids 267-283 of SEQ ID NO:2 has amolecular weight of approximately 1,989 Daltons. The peptide encoded byamino acids 292-305 of SEQ ID NO:2 has a molecular weight ofapproximately 1,757 Daltons. The peptide encoded by amino acids 313-333of SEQ ID NO:2 has a molecular weight of approximately 2,601 Daltons.The peptide encoded by amino acids 334-353 of SEQ ID NO:2 has amolecular weight of approximately 2,324 Daltons. The peptide encoded byamino acids 355-395 of SEQ ID NO:2 has a molecular weight ofapproximately 4,765 Daltons. The peptide encoded by amino acids 406-471of SEQ ID NO:2 has a molecular weight of approximately 7,339 Daltons.The peptide encoded by amino acids 473-507 of SEQ ID NO:2 has amolecular weight of approximately 3,885 Daltons. The peptide encoded byamino acids 513-527 of SEQ ID NO:2 has a molecular weight ofapproximately 1,785 Daltons. The peptide encoded by amino acids 529-539of SEQ ID NO:2 has a molecular weight of approximately 1,282 Daltons.The peptide encoded by amino acids 543-561 of SEQ ID NO:2 has amolecular weight of approximately 2,329 Daltons. The peptide encoded byamino acids 562-576 of SEQ ID NO:2 has a molecular weight ofapproximately 1,855 Daltons. The peptide encoded by amino acids 596-612of SEQ ID NO:2 has a molecular weight of approximately 1,858 Daltons.

[0214] Therefore, cleavage of the mouse ACPL polypeptide by enzymatictreatment with Achromobacter protease I generates a unique set of ACPLfragmented peptide molecular weight markers. The unique and known aminoacid sequence of these fragmented peptides allows the determination ofthe molecular weight of these ACPL fragmented peptide molecular weightmarkers. In this particular case, these ACPL fragmented peptidemolecular weight markers have molecular weights of approximately 1,897;1,236; 3,100; 1,721; 7,285; 4,893; 4,123; 1,866; 1,989; 1,757; 2,601;2,324; 4,765; 7,339; 3,885; 1,785; 1,282; 2,329; 1,855; and 1,858Daltons.

[0215] Once again, the ACPL fragmented peptide molecular weight markers,together with a sample protein, can be resolved by denaturingpolyacrylamide gel electrophoresis by conventional means in two separatelanes of a gel containing sodium dodecyl sulfate and a concentration ofacrylamide between 10-20%. Proteins on the gel can be visualized using aconventional staining procedure. The ACPL fragmented peptide molecularweight markers can be used as molecular weight markers in the estimationof the apparent molecular weight of the sample protein. The ACPLfragmented peptide molecular weight markers serve particularly well asmolecular weight markers for the estimation of the apparent molecularweight of proteins that have apparent molecular weights close to 1,897;1,236; 3,100; 1,721; 7,285; 4,893; 4,123; 1,866; 1,989; 1,757; 2,601;2,324; 4,765; 7,339; 3,885; 1,785; 1,282; 2,329; 1,855; or 1,858Daltons. The use of these fragmented peptide molecular weight markersallows an increased accuracy in the determination of apparent molecularweight of proteins that have apparent molecular weights close to 1,897;1,236; 3,100; 1,721; 7,285; 4,893; 4,123; 1,866; 1,989; 1,757; 2,601;2,324; 4,765; 7,339; 3,885; 1,785; 1,282; 2,329; 1,855; or 1,858Daltons. It is understood of course that the unique amino acid sequenceof human ACPL polypeptide (SEQ ID NO:13) can similarly be used togenerate a unique set of fragmented peptide molecular weight markers.The fragment sizes can readily be determined using available computerprograms.

[0216] In another embodiment, the sample protein and the ACPLpolypeptide can be simultaneously, but separately, treated withAchromobacter protease I under conventional conditions that result infragmentation of the sample protein and the ACPL polypeptide by specifichydrolysis on the carboxyl side of the lysine residues within the sampleprotein and the ACPL polypeptide. The ACPL fragmented peptide molecularweight markers and the fragmented peptides derived from the sampleprotein are resolved by denaturing polyacrylamide gel electrophoresis byconventional means in two separate lanes of a gel containing sodiumdodecyl sulfate and a concentration of acrylamide between 10-20%.Fragmented peptides on the gel can be visualized using a conventionalstaining procedure. The ACPL fragmented peptide molecular weight markerscan be used as molecular weight markers in the estimation of theapparent molecular weight of the sample protein. The ACPL fragmentedpeptide molecular weight markers serve particularly well as molecularweight markers for the estimation of the apparent molecular weight offragmented peptides that have apparent molecular weights close to 1,897;1,236; 3,100; 1,721; 7,285; 4,893; 4,123; 1,866; 1,989; 1,757; 2,601;2,324; 4,765; 7,339; 3,885; 1,785; 1,282; 2,329; 1,855; or 1,858Daltons. The use of these ACPL fragmented peptide molecular weightmarkers allows an increased accuracy in the determination of apparentmolecular weight of fragmented peptides that have apparent molecularweights close to 1,897; 1,236; 3,100; 1,721; 7,285; 4,893; 4,123; 1,866;1,989; 1,757; 2,601; 2,324; 4,765; 7,339; 3,885; 1,785; 1,282; 2,329;1,855; or 1,858 Daltons. The extent of fragmentation of the ACPLpolypeptide is further used as a control to determine the conditionsexpected for complete fragmentation of the sample protein. It isunderstood of course that many enzymes could be used to fragment ACPLpolypeptides and that this embodiment in no way limits the scope of theinvention.

[0217] In another embodiment, monoclonal and polyclonal antibodiesagainst ACPL polypeptides can be generated. Balb/c mice can be injectedintraperitoneally on two occasions at 3 week intervals with 10 μg ofisolated and purified ACPL polypeptide or peptides based on the aminoacid sequence of ACPL polypeptides in the presence of RIBI adjuvant(RIBI Corp., Hamilton, Mont.). Mouse sera are then assayed byconventional dot blot technique or antibody capture (ABC) to determinewhich animal is best to fuse. Three weeks later, mice are given anintravenous boost of 3 μg of the ACPL polypeptide or peptides, suspendedin sterile PBS. Three days later, mice are sacrificed and spleen cellsfused with Ag8.653 myeloma cells (ATCC) following established protocols.Briefly, Ag8.653 cells are washed several times in serum-free media andfused to mouse spleen cells at a ratio of three spleen cells to onemyeloma cell. The fusing agent is 50% PEG: 10% DMSO (Sigma). Fusion isplated out into twenty 96-well flat bottom plates (Corning) containingHAT supplemented DMEM media and allowed to grow for eight days.Supernatants from resultant hybridomas are collected and added to a96-well plate for 60 minutes that is first coated with goat anti-mouseIg. Following washes, ¹²⁵I-ACPL polypeptide or peptides are added toeach well, incubated for 60 minutes at room temperature, and washed fourtimes. Positive wells can be subsequently detected by autoradiography at−70 C using Kodak X-Omat S film. Positive clones can be grown in bulkculture and supernatants are subsequently purified over a Protein Acolumn (Pharmacia). It is understood of course that many techniquescould be used to generate antibodies against ACPL polypeptides andfragmented peptides thereof and that this embodiment in no way limitsthe scope of the invention.

[0218] In another embodiment, antibodies generated against ACPLpolypeptides and fragmented peptides thereof can be used in combinationwith ACPL polypeptide or fragmented peptide molecular weight markers toenhance the accuracy in the use of these molecular weight markers todetermine the apparent molecular weight and isoelectric point of asample protein. ACPL polypeptide or fragmented peptide molecular weightmarkers can be mixed with a molar excess of a sample protein and themixture can be resolved by two dimensional electrophoresis byconventional means. Polypeptides can be transferred to a suitableprotein binding membrane, such as nitrocellulose, by conventional means.

[0219] Polypeptides on the membrane can be visualized using twodifferent methods that allow a discrimination between the sample proteinand the molecular weight markers. ACPL polypeptide or fragmented peptidemolecular weight markers can be visualized using antibodies generatedagainst these markers and conventional immunoblotting techniques. Thisdetection is performed under conventional conditions that do not resultin the detection of the sample protein. It is understood that it may notbe possible to generate antibodies against all ACPL polypeptidefragments, since small peptides may not contain immunogenic epitopes. Itis further understood that not all antibodies will work in this assay;however, those antibodies which are able to bind ACPL polypeptides andfragments can be readily determined using conventional techniques.

[0220] The sample protein is visualized using a conventional stainingprocedure. The molar excess of sample protein to ACPL polypeptide orfragmented peptide molecular weight markers is such that theconventional staining procedure predominantly detects the sampleprotein. The level of ACPL polypeptide or fragmented peptide molecularweight markers is such as to allow little or no detection of thesemarkers by the conventional staining method. The preferred molar excessof sample protein to ACPL polypeptide molecular weight markers isbetween 2 and 100,000 fold. More preferably, the preferred molar excessof sample protein to ACPL polypeptide molecular weight markers isbetween 10 and 10,000 fold and especially between 100 and 1,000 fold.

[0221] The ACPL polypeptide or fragmented peptide molecular weightmarkers can be used as molecular weight and isoelectric point markers inthe estimation of the apparent molecular weight and isoelectric point ofthe sample protein. The ACPL polypeptide or fragmented peptide molecularweight markers serve particularly well as molecular weight andisoelectric point markers for the estimation of apparent molecularweights and isoelectric points of sample proteins that have apparentmolecular weights and isoelectric points close to that of the ACPLpolypeptide or fragmented peptide molecular weight markers. The abilityto simultaneously resolve the ACPL polypeptide or fragmented peptidemolecular weight markers and the sample protein under identicalconditions allows for increased accuracy in the determination of theapparent molecular weight and isoelectric point of the sample protein.This is of particular interest in techniques, such as two dimensionalelectrophoresis, where the nature of the procedure dictates that anymarkers should be resolved simultaneously with the sample protein.

[0222] In another embodiment, ACPL polypeptide or fragmented peptidemolecular weight markers can be used as molecular weight and isoelectricpoint markers in the estimation of the apparent molecular weight andisoelectric point of fragmented peptides derived by treatment of asample protein with a cleavage agent. It is understood that manytechniques can be used for the determination of molecular weight andisoelectric point of a sample protein and fragmented peptides thereofusing ACPL polypeptide molecular weight markers and peptide fragmentsthereof and that this embodiment in no way limits the scope of theinvention.

[0223] ACPL polypeptide molecular weight markers encompassed byinvention can have variable molecular weights, depending upon the hostcell in which they are expressed. Glycosylation of ACPL polypeptidemolecular weight markers and peptide fragments thereof in various celltypes can result in variations of the molecular weight of these markers,depending upon the extent of modification. The size of ACPL polypeptidemolecular weight markers can be most heterogeneous with fragments ofACPL polypeptide derived from the extracellular portion of thepolypeptide. Consistent molecular weight markers can be obtained byusing polypeptides derived entirely from the transmembrane andcytoplasmic regions, pretreating with N-glycanase to removeglycosylation, or expressing the polypeptides in bacterial hosts.

[0224] The polypeptides of the present invention can be subjected tofragmentation into smaller peptides by chemical and enzymatic means, andthe peptide fragments so produced can be used in the analysis of otherproteins or polypeptides. For example, such peptide fragments can beused as peptide molecular weight markers, peptide isoelectric pointmarkers, or in the analysis of the degree of peptide fragmentation.Thus, the invention also includes these polypeptides and peptidefragments, as well as kits to aid in the determination of the apparentmolecular weight and isoelectric point of an unknown protein and kits toassess the degree of fragmentation of an unknown protein.

[0225] Of course, the peptides and fragments of the polypeptides of theinvention can also be produced by conventional recombinant processes andsynthetic processes well known in the art. With regard to recombinantprocesses, the polypeptides and peptide fragments encompassed byinvention can have variable molecular weights, depending upon the hostcell in which they are expressed. Glycosylation of polypeptides andpeptide fragments of the invention in various cell types can result invariations of the molecular weight of these pieces, depending upon theextent of modification. The size of these pieces can be mostheterogeneous with fragments of polypeptide derived from theextracellular portion of the polypeptide. Consistent polypeptides andpeptide fragments can be obtained by using polypeptides derived entirelyfrom the transmembrane and cytoplasmic regions, pretreating withN-glycanase to remove glycosylation, or expressing the polypeptides inbacterial hosts.

[0226] The molecular weight of these polypeptides can also be varied byfusing additional peptide sequences to both the amino and carboxylterminal ends of polypeptides of the invention. Fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention can be used to enhance expression of thesepolypeptides or aid in the purification of the protein. In addition,fusions of additional peptide sequences at the amino and carboxylterminal ends of polypeptides of the invention will alter some, butusually not all, of the fragmented peptides of the polypeptidesgenerated by enzymatic or chemical treatment. Of course, mutations canbe introduced into polypeptides of the invention using routine and knowntechniques of molecular biology. For example, a mutation can be designedso as to eliminate a site of proteolytic cleavage by a specific enzymeor a site of cleavage by a specific chemically induced fragmentationprocedure. The elimination of the site will alter the peptidefingerprint of polypeptides of the invention upon fragmentation with thespecific enzyme or chemical procedure.

[0227] The polypeptides and the resultant fragmented peptides can beanalyzed by methods including sedimentation, electrophoresis,chromatography, and mass spectrometry to determine their molecularweights. Because the unique amino acid sequence of each piece specifiesa molecular weight, these pieces can thereafter serve as molecularweight markers using such analysis techniques to assist in thedetermination of the molecular weight of an unknown protein,polypeptides or fragments thereof. The molecular weight markers of theinvention serve particularly well as molecular weight markers for theestimation of the apparent molecular weight of proteins that havesimilar apparent molecular weights and, consequently, allow increasedaccuracy in the determination of apparent molecular weight of proteins.

[0228] When the invention relates to the use of fragmented peptidemolecular weight markers, those markers are preferably at least 10 aminoacids in size. More preferably, these fragmented peptide molecularweight markers are between 10 and 100 amino acids in size. Even morepreferable are fragmented peptide molecular weight markers between 10and 50 amino acids in size and especially between 10 and 35 amino acidsin size. Most preferable are fragmented peptide molecular weight markersbetween 10 and 20 amino acids in size.

[0229] Among the methods for determining molecular weight aresedimentation, gel electrophoresis, chromatography, and massspectrometry. A particularly preferred embodiment is denaturingpolyacrylamide gel electrophoresis (U. K. Laemmli, Nature 227:680-685,1970). Conventionally, the method uses two separate lanes of a gelcontaining sodium dodecyl sulfate and a concentration of acrylamidebetween 6-20%. The ability to simultaneously resolve the marker and thesample under identical conditions allows for increased accuracy. It isunderstood, of course, that many different techniques can be used forthe determination of the molecular weight of an unknown protein usingpolypeptides of the invention, and that this embodiment in no way limitsthe scope of the invention.

[0230] Each unglycosylated polypeptide or fragment thereof has a pI thatis intrinsically determined by its unique amino acid sequence (which pIcan be estimated by the skilled artisan using any of the computerprograms designed to predict pI values currently available, calculatedusing any well-known amino acid pKa table, or measured empirically).Therefore these polypeptides and fragments thereof can serve as specificmarkers to assist in the determination of the isoelectric point of anunknown protein, polypeptide, or fragmented peptide using techniquessuch as isoelectric focusing. These polypeptide or fragmented peptidemarkers serve particularly well for the estimation of apparentisoelectric points of unknown proteins that have apparent isoelectricpoints close to that of the polypeptide or fragmented peptide markers ofthe invention.

[0231] The technique of isoelectric focusing can be further combinedwith other techniques such as gel electrophoresis to simultaneouslyseparate a protein on the basis of molecular weight and charge. Theability to simultaneously resolve these polypeptide or fragmentedpeptide markers and the unknown protein under identical conditionsallows for increased accuracy in the determination of the apparentisoelectric point of the unknown protein. This is of particular interestin techniques, such as two dimensional electrophoresis (T. D. Brock andM. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed.1991)), where the nature of the procedure dictates that any markersshould be resolved simultaneously with the unknown protein. In addition,with such methods, these polypeptides and fragmented peptides thereofcan assist in the determination of both the isoelectric point andmolecular weight of an unknown protein or fragmented peptide.

[0232] Polypeptides and fragmented peptides can be visualized using twodifferent methods that allow a discrimination between the unknownprotein and the molecular weight markers. In one embodiment, thepolypeptide and fragmented peptide molecular weight markers of theinvention can be visualized using antibodies generated against thesemarkers and conventional immunoblotting techniques. This detection isperformed under conventional conditions that do not result in thedetection of the unknown protein. It is understood that it may not bepossible to generate antibodies against all polypeptide fragments of theinvention, since small peptides may not contain immunogenic epitopes. Itis further understood that not all antibodies will work in this assay;however, those antibodies which are able to bind polypeptides andfragments of the invention can be readily determined using conventionaltechniques.

[0233] The unknown protein is also visualized by using a conventionalstaining procedure. The molar excess of unknown protein to polypeptideor fragmented peptide molecular weight markers of the invention is suchthat the conventional staining procedure predominantly detects theunknown protein. The level of these polypeptide or fragmented peptidemolecular weight markers is such as to allow little or no detection ofthese markers by the conventional staining method. The preferred molarexcess of unknown protein to polypeptide molecular weight markers of theinvention is between 2 and 100,000 fold. More preferably, the preferredmolar excess of unknown protein to these polypeptide molecular weightmarkers is between 10 and 10,000 fold and especially between 100 and1,000 fold.

[0234] It is understood of course that many techniques can be used forthe determination and detection of molecular weight and isoelectricpoint of an unknown protein, polypeptides, and fragmented peptidesthereof using these polypeptide molecular weight markers and peptidefragments thereof and that these embodiments in no way limit the scopeof the invention.

[0235] In another embodiment, the analysis of the progressivefragmentation of the polypeptides of the invention into specificpeptides (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977),such as by altering the time or temperature of the fragmentationreaction, can be used as a control for the extent of cleavage of anunknown protein. For example, cleavage of the same amount of polypeptideand unknown protein under identical conditions can allow for a directcomparison of the extent of fragmentation. Conditions that result in thecomplete fragmentation of the polypeptide can also result in completefragmentation of the unknown protein.

[0236] Finally, as to the kits that are encompassed by the invention,the constituents of such kits can be varied, but typically contain thepolypeptide and fragmented peptide molecular weight markers. Also, suchkits can contain the polypeptides wherein a site necessary forfragmentation has been removed. Furthermore, the kits can containreagents for the specific cleavage of the polypeptide and the unknownprotein by chemical or enzymatic cleavage. Kits can further containantibodies directed against polypeptides or fragments thereof of theinvention.

[0237] Identification of Unknown Proteins

[0238] As set forth above, a polypeptide or peptide fingerprint can beentered into or compared to a database of known proteins to assist inthe identification of the unknown protein using mass spectrometry (W. J.Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyoet al., Electrophoresis 19:998-1005, 1998). A variety of computersoftware programs to facilitate these comparisons are accessible via theInternet, such as Protein Prospector (Internet site:prospector.uscf.edu), MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internetsite:www.mann.embl-heiedelberg.de . . . deSearch/FR_PeptideSearchForm.html), and ProFound (Internetsite:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). Theseprograms allow the user to specify the cleavage agent and the molecularweights of the fragmented peptides within a designated tolerance. Theprograms compare observed molecular weights to predicted peptidemolecular weights derived from sequence databases to assist indetermining the identity of the unknown protein.

[0239] In addition, a polypeptide or peptide digest can be sequencedusing tandem mass spectrometry (MS/MS) and the resulting sequencesearched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec.5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994);J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec.11:1067-1075(1997)). Searching programs that can be used in this process exist onthe Internet, such as Lutefisk 97 (Internet site:www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, PeptideSearch and ProFound programs described above.

[0240] Therefore, adding the sequence of a gene and its predictedprotein sequence and peptide fragments to a sequence database can aid inthe identification of unknown proteins using mass spectrometry.

[0241] Antibodies

[0242] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0243] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

[0244] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0245] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

[0246] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

[0247] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0248] Antigen-binding fragments of the antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab and F(ab′)₂ fragments. Antibody fragments and derivatives producedby genetic engineering techniques are also provided.

[0249] In one embodiment, the antibodies are specific for thepolypeptides of the present invention and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0250] Uses Thereof

[0251] The antibodies of the invention can be used in assays to detectthe presence of the polypeptides or fragments of the invention, eitherin vitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

[0252] Those antibodies that additionally can block binding of thepolypeptides of the invention to its binding partner may be used toinhibit a biological activity that results from such binding. Suchblocking antibodies may be identified using any suitable assayprocedure, such as by testing antibodies for the ability to inhibitbinding of ACPL to certain cells expressing the binding partner.Alternatively, blocking antibodies may be identified in assays for theability to inhibit a biological effect that results from binding of anACPL binding partner to target cells. Antibodies may be assayed for theability to inhibit binding partner-mediated activity.

[0253] Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of an ACPL binding partnerwith cell surface binding partner receptor thus may be treated. Atherapeutic method involves in vivo administration of a blockingantibody to a mammal in an amount effective in inhibiting a bindingpartner-mediated biological activity. Monoclonal antibodies aregenerally preferred for use in such therapeutic methods. In oneembodiment, an antigen-binding antibody fragment is employed.

[0254] Antibodies may be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to cell surface bindingpartner, induce biological effects (e.g., transduction of biologicalsignals) similar to the biological effects induced when an ACPL bindingpartner binds to cell surface ACPL. Agonistic antibodies may be used toinduce IL-18 mediated activity.

[0255] Compositions comprising an antibody that is directed againstACPL, and a physiologically acceptable diluent, excipient, or carrier,are provided herein. Suitable components of such compositions are asdescribed above for compositions containing ACPL proteins.

[0256] Also provided herein are conjugates comprising a detectable(e.g., diagnostic) or therapeutic agent, attached to the antibody.Examples of such agents are presented above. The conjugates find use inin vitro or in vivo procedures.

[0257] The following examples are provided to further illustrateparticular embodiments of the invention, and are not to be construed aslimiting the scope of the present invention. The specification is mostthoroughly understood in light of the teachings of the references citedwithin the specification, which are hereby incorporated by reference.The embodiments within the specification and the following Examplesprovide an illustration of embodiments of the invention and should notbe construed to limit the scope of the invention. The skilled artisanrecognizes many other embodiments are encompassed by the claimedinvention. In the following Examples, all methods described areconventional unless otherwise specified.

EXAMPLE 1 Isolation of the Mouse Nucleic Acid

[0258] A mouse ACPL cDNA was isolated by searching the expressedsequence tag data base discovering the IMAGE clone 640615 (GenBankAccession number AA203097) has homology to IL-1RAcP. IMAGE clone 640615was obtained, labeled with ³²P by random priming and used to probe anEL46.1 (mouse thymocyte) cDNA library. The hybridization was carried outat 42° C. in hybridization solution containing 50% formamide. After thefull-length open reading frame was defined by an EL46.1cDNA clone, itwas verified by obtaining independent isolates from 7B9 (mouse T cell)and LDA 11 (mouse bone marrow stromal) cDNA libraries using PCRamplification. Primers corresponded to nucleotides −15 to +13 andnucleotides 1892 to 1916 (relative to the initiating ATG being +1 to +3)of the mouse ACPL nucleotide sequence.

EXAMPLE 2 Isolation of Human ACPL Nucleic Acid Sequence

[0259] A human cDNA clone, termed QQ1352, obtained by random sequencingof an NK cell library, was found to have a high degree of homology tothe murine ACPL isolated as described in Example 1. The clone was usedas a probe to isolate human ACPL clones from peripheral bloodlymphocyte, peripheral blood T cell, and NK cDNA libraries. The regionof clone QQ1352 that was used as a probe was homologous to mouse ACPLnucleotides 1196-11753. A full-length c lone was not obtained from anyof the libraries so vector-anchored PCR was carried out in each of thelibraries to obtain the 5′ end of the open reading frame (SEQ ID NO:6)

EXAMPLE 3 Determining Chromosome Map Position

[0260] The chromosome map position of human ACPL was determined byradiation hybrid mapping using the Stanford G3 Radiation Hybrid Panel(Research Genetics). The primers were selected for their homology toIL-1R. Amplification was carried out under standard PCR conditions for40 cycles.

[0261] The results placed human ACPL on chromosome 2, most closelylinked to AFM316tg5, with a logarithm of odds score of 12.72. This isthe same region of chromosome 2 to which IL-1R type I, Il-1R type II,IL-1R-rp1, and TI/ST2 have been mapped.

EXAMPLE 4 Northern Blot Analysis

[0262] A human multiple tissue blot was purchased from CLONTECHlaboratories, Inc. and contained 2 μg of mRNA from normal human spleen,thymus, prostate, testis, ovary, small intestine, colon, and peripheralblood leukocyte. This was hybridized overnight with a ³²P-labeledantisense human ACPL riboprobe in hybridization buffer containing 50%formamide at 63° C. and then washed at 68° C. in 0.1×SSC/0.1% SDS. Afterexposure, the blot was rehybridized with a random-prime labeled probeagainst β-actin for standardization.

[0263] The results demonstrated that human ACPL is expressed strongly inperipheral blood leukocytes and spleen. To a less extent, human ACPL isexpressed in colon. The results further demonstrated that human ACPL isweakly expressed in prostate and small intestine mRNA. The predominantmRNA product was approximately 3.8 kilobases, with minor bnds atapproximately 2.6 and 8.0 kilobases. Expression was also detected inlung mRNA by Northern analysis.

EXAMPLE 5 Expressing ACPL Polypeptide and ACPL:FC Fusions

[0264] To express mouse and human ACPL polypeptides, full-length mouseand human ACPL nucleotide sequences were generated by PCR and clonedinto pDC304, a variant of pDC302. For expressing human ACPL:Fc fusionprotein, the extracellular portion of the human ACPL expression vector(amino acids 1-356) was joined to the CH2 and CH3 domains of human IgG1and was generated as described in Baum et al, EMBO J. 13:39924001(1994).

EXAMPLE 6 Induction of NFkB through ACPL Polypeptide in COS and S49Cells

[0265] In order to determine whether mouse ACPL polypeptide (SEQ IDNO:2) was a receptor involved in IL-18 signaling, ACPL polypeptide wasoverexpressed in COS cells and S49.1 cells, and the effect of IL-18stimulation on NFkB activation was assessed. COS-7 cells weretransfected by the DEAE/Dextran method in a 12-well format. Each wellwas transfected with a total of 200 ng of expression vectors encodingreceptor(s) and 800 ng of a NFkB-Luc reporter plasmid, which contains 3NFkB sites mediating luciferase expression. Approximately 10⁷ S49.1cells were transfected by electroporation in 0.7 ml with 40 μg of theNFkB-Luc reporter plasmid, and a total of 20 μg of expression vectorsencoding receptors. Electroporations were performed at 960 μF and 320V.

[0266] Cells were incubated for 2 days, and then stimulated with 40ng/ml murine IL-18 (PeproTech) for 4 hours. Cells were washed, lysed,and assayed for luciferase activity using Luciferase Assay Reagents(Promega Corp.) according to the manufacturer's instructions.

[0267] Cells transfected with vector alone, expression vector encodingIL-1Rrp1 alone, or expression vector encoding ACPL polypeptide alonewere not responsive to IL-18 stimulation. Furthermore, no function ofACPL in IL-1 signaling was detected when the expression vector encodingthe receptor was transfected alone, or in combination with an expressionvector encoding IL-1R type 1 or IL-1RAcP. However, the addition of IL-18to cells cotransfected with expression vectors encoding IL-1Rrp1 andACPL polypeptide induced expression of the NFkB-linked gene 10-fold inCOS cells and 300-fold in S49 cells. This dramatic stimulation of NFkBactivity indicates that ACPL polypeptide is a component of the IL-18receptor that cooperates synergistically with IL-1Rrp1 to induce NFkBsignaling in response to IL-18 stimulation.

EXAMPLE 7 Activation of JNK Activity

[0268] The induction of JNK activity is a downstream signaling event inthe IL-1 pathway. Similar to the induction of NFkB experiment above, itwas examined whether ACPL alone or in combination with IL-1Rrp1 wascapable of mediating the indcution of JNK activity by IL-18. Activationof JNK activity was assessed as described in Bird et al. J. Biol. Chem.269:31836-31844, 1994. Two (2) days post-transfection, COS7 cells weresimulated with IL-18 for 15 minutes, lysed, and immunoprecipitated witha combination of two anti-JNK antibodies (c-17 and FL, Santa CruzBiotaechnology, Inc). This immunocomplex was assayed for activity byaddition of glutathione S-transferase-c-Jun (Upstate Biotechnology,Inc.) and [γ-³²]ATP in kinase buffer. The reaction was allowed toproceed for 30 minutes at room temperature, after which Laemmli Loadingbuffer was added to stop the reaction, and products were electrophoresedon a 4-20% acrylamide getl, stained, dried, and analyzed on aPhosphorImager.

[0269] Similar to the results obtained regarding activation of NFκB, JNKactivity was induced only by IL-18 in COS7 cells when IL-1R-Rp1 and ACPLwere coexpressed.

1 7 1 1845 DNA Mus sp. CDS (1)..(1845) 1 atg ctc tgt ttg ggc tgg gtg tttctt tgg ttt gtt gca gga gag aag 48 Met Leu Cys Leu Gly Trp Val Phe LeuTrp Phe Val Ala Gly Glu Lys 1 5 10 15 acc aca gga ttt aat cat tca gcttgt gcc acc aaa aaa ctt ctg tgg 96 Thr Thr Gly Phe Asn His Ser Ala CysAla Thr Lys Lys Leu Leu Trp 20 25 30 aca tat tct gca agg ggt gca gag aatttt gtc cta ttt tgt gac tta 144 Thr Tyr Ser Ala Arg Gly Ala Glu Asn PheVal Leu Phe Cys Asp Leu 35 40 45 caa gag ctt cag gag caa aaa ttc tcc catgca agt caa ctg tca cca 192 Gln Glu Leu Gln Glu Gln Lys Phe Ser His AlaSer Gln Leu Ser Pro 50 55 60 aca caa agt cct gct cac aaa cct tgc agt ggcagt cag aag gac cta 240 Thr Gln Ser Pro Ala His Lys Pro Cys Ser Gly SerGln Lys Asp Leu 65 70 75 80 tct gat gtc cag tgg tac atg caa cct cgg agtgga agt cca cta gag 288 Ser Asp Val Gln Trp Tyr Met Gln Pro Arg Ser GlySer Pro Leu Glu 85 90 95 gag atc agt aga aac tct ccc cat atg cag agt gaaggc atg ctg cat 336 Glu Ile Ser Arg Asn Ser Pro His Met Gln Ser Glu GlyMet Leu His 100 105 110 ata ttg gcc cca cag acg aac agc att tgg tca tatatt tgt aga ccc 384 Ile Leu Ala Pro Gln Thr Asn Ser Ile Trp Ser Tyr IleCys Arg Pro 115 120 125 aga att agg agc ccc cag gat atg gcc tgt tgt atcaag aca gtc tta 432 Arg Ile Arg Ser Pro Gln Asp Met Ala Cys Cys Ile LysThr Val Leu 130 135 140 gaa gtt aag cct cag aga aac gtg tcc tgt ggg aacaca gca caa gat 480 Glu Val Lys Pro Gln Arg Asn Val Ser Cys Gly Asn ThrAla Gln Asp 145 150 155 160 gaa caa gtc cta ctt ctt ggc agt act ggc tccatt cat tgt ccc agt 528 Glu Gln Val Leu Leu Leu Gly Ser Thr Gly Ser IleHis Cys Pro Ser 165 170 175 ctc agc tgc caa agt gat gta cag agt cca gagatg acc tgg tac aag 576 Leu Ser Cys Gln Ser Asp Val Gln Ser Pro Glu MetThr Trp Tyr Lys 180 185 190 gat gga aga cta ctt cct gag cac aag aaa aatcca att gag atg gca 624 Asp Gly Arg Leu Leu Pro Glu His Lys Lys Asn ProIle Glu Met Ala 195 200 205 gat att tat gtt ttt aat caa ggc ttg tat gtatgt gat tac aca cag 672 Asp Ile Tyr Val Phe Asn Gln Gly Leu Tyr Val CysAsp Tyr Thr Gln 210 215 220 tca gat aat gtg agt tcc tgg aca gtc cga gctgtg gtt aaa gtg aga 720 Ser Asp Asn Val Ser Ser Trp Thr Val Arg Ala ValVal Lys Val Arg 225 230 235 240 acc att ggt aag gac atc aat gtg aag ccggaa att ctg gat ccc att 768 Thr Ile Gly Lys Asp Ile Asn Val Lys Pro GluIle Leu Asp Pro Ile 245 250 255 aca gat aca ctg gac gta gag ctt gga aagcct tta act ctc ccc tgc 816 Thr Asp Thr Leu Asp Val Glu Leu Gly Lys ProLeu Thr Leu Pro Cys 260 265 270 aga gta cag ttt ggc ttc caa aga ctt tcaaag cct gtg ata aag tgg 864 Arg Val Gln Phe Gly Phe Gln Arg Leu Ser LysPro Val Ile Lys Trp 275 280 285 tat gtc aaa gaa tct aca cag gag tgg gaaatg tca gta ttt gag gag 912 Tyr Val Lys Glu Ser Thr Gln Glu Trp Glu MetSer Val Phe Glu Glu 290 295 300 aaa aga att caa tcc act ttc aag aat gaagtc att gaa cgt acc atc 960 Lys Arg Ile Gln Ser Thr Phe Lys Asn Glu ValIle Glu Arg Thr Ile 305 310 315 320 ttc ttg aga gaa gtt acc cag aga gatctc agc aga aag ttt gtt tgc 1008 Phe Leu Arg Glu Val Thr Gln Arg Asp LeuSer Arg Lys Phe Val Cys 325 330 335 ttt gcc cag aac tcc att ggg aac acaaca cgg acc ata cgg ctg agg 1056 Phe Ala Gln Asn Ser Ile Gly Asn Thr ThrArg Thr Ile Arg Leu Arg 340 345 350 aag aag gaa gag gtg gtg ttt gta tacatc ctt ctc ggc acg gcc ttg 1104 Lys Lys Glu Glu Val Val Phe Val Tyr IleLeu Leu Gly Thr Ala Leu 355 360 365 atg ctg gtg ggc gtt ctg gtg gca gctgct ttc ctc tac tgg tac tgg 1152 Met Leu Val Gly Val Leu Val Ala Ala AlaPhe Leu Tyr Trp Tyr Trp 370 375 380 att gaa gtt gtc ctg ctc tgt cga acctac aag aac aaa gat gag act 1200 Ile Glu Val Val Leu Leu Cys Arg Thr TyrLys Asn Lys Asp Glu Thr 385 390 395 400 ctg ggg gat aag aag gaa ttc gatgca ttt gta tcc tac tcg aat tgg 1248 Leu Gly Asp Lys Lys Glu Phe Asp AlaPhe Val Ser Tyr Ser Asn Trp 405 410 415 agc tct cct gag act gac gcc gtggga tct ctg agt gag gaa cac ctg 1296 Ser Ser Pro Glu Thr Asp Ala Val GlySer Leu Ser Glu Glu His Leu 420 425 430 gct ctg aat ctt ttc ccg gaa gtgcta gaa gac acc tat ggg tac aga 1344 Ala Leu Asn Leu Phe Pro Glu Val LeuGlu Asp Thr Tyr Gly Tyr Arg 435 440 445 ttg tgt ttg ctt gac cga gat gtgacc cca gga gga gtg tat gca gat 1392 Leu Cys Leu Leu Asp Arg Asp Val ThrPro Gly Gly Val Tyr Ala Asp 450 455 460 gac att gtg agc atc att aag aaaagc cga aga gga ata ttt atc ctg 1440 Asp Ile Val Ser Ile Ile Lys Lys SerArg Arg Gly Ile Phe Ile Leu 465 470 475 480 agt ccc agc tac ctc aat ggaccc cgt gtc ttt gag cta caa gca gca 1488 Ser Pro Ser Tyr Leu Asn Gly ProArg Val Phe Glu Leu Gln Ala Ala 485 490 495 gtg aat ctt gcc ttg gtt gatcag aca ctg aag ttg att tta att aag 1536 Val Asn Leu Ala Leu Val Asp GlnThr Leu Lys Leu Ile Leu Ile Lys 500 505 510 ttc tgt tcc ttc caa gag ccagaa tct ctt cct tac ctt gtc aaa aag 1584 Phe Cys Ser Phe Gln Glu Pro GluSer Leu Pro Tyr Leu Val Lys Lys 515 520 525 gct ctg cgg gtt ctc ccc acagtc aca tgg aaa ggc ttg aag tcg gtc 1632 Ala Leu Arg Val Leu Pro Thr ValThr Trp Lys Gly Leu Lys Ser Val 530 535 540 cac gcc agt tcc agg ttc tggacc caa att cgt tac cac atg cct gtg 1680 His Ala Ser Ser Arg Phe Trp ThrGln Ile Arg Tyr His Met Pro Val 545 550 555 560 aag aac tcc aac agg tttatg ttc aac ggg ctc aga att ttc ctg aag 1728 Lys Asn Ser Asn Arg Phe MetPhe Asn Gly Leu Arg Ile Phe Leu Lys 565 570 575 ggc ttt tcc cct gaa aaggac cta gtg aca cag aaa ccc ctg gaa gga 1776 Gly Phe Ser Pro Glu Lys AspLeu Val Thr Gln Lys Pro Leu Glu Gly 580 585 590 atg ccc aag tct ggg aatgac cac gga gct cag aac ctc ctt ctc tac 1824 Met Pro Lys Ser Gly Asn AspHis Gly Ala Gln Asn Leu Leu Leu Tyr 595 600 605 agt gac cag aag agg tgctga 1845 Ser Asp Gln Lys Arg Cys 610 2 614 PRT Mus sp. 2 Met Leu Cys LeuGly Trp Val Phe Leu Trp Phe Val Ala Gly Glu Lys 1 5 10 15 Thr Thr GlyPhe Asn His Ser Ala Cys Ala Thr Lys Lys Leu Leu Trp 20 25 30 Thr Tyr SerAla Arg Gly Ala Glu Asn Phe Val Leu Phe Cys Asp Leu 35 40 45 Gln Glu LeuGln Glu Gln Lys Phe Ser His Ala Ser Gln Leu Ser Pro 50 55 60 Thr Gln SerPro Ala His Lys Pro Cys Ser Gly Ser Gln Lys Asp Leu 65 70 75 80 Ser AspVal Gln Trp Tyr Met Gln Pro Arg Ser Gly Ser Pro Leu Glu 85 90 95 Glu IleSer Arg Asn Ser Pro His Met Gln Ser Glu Gly Met Leu His 100 105 110 IleLeu Ala Pro Gln Thr Asn Ser Ile Trp Ser Tyr Ile Cys Arg Pro 115 120 125Arg Ile Arg Ser Pro Gln Asp Met Ala Cys Cys Ile Lys Thr Val Leu 130 135140 Glu Val Lys Pro Gln Arg Asn Val Ser Cys Gly Asn Thr Ala Gln Asp 145150 155 160 Glu Gln Val Leu Leu Leu Gly Ser Thr Gly Ser Ile His Cys ProSer 165 170 175 Leu Ser Cys Gln Ser Asp Val Gln Ser Pro Glu Met Thr TrpTyr Lys 180 185 190 Asp Gly Arg Leu Leu Pro Glu His Lys Lys Asn Pro IleGlu Met Ala 195 200 205 Asp Ile Tyr Val Phe Asn Gln Gly Leu Tyr Val CysAsp Tyr Thr Gln 210 215 220 Ser Asp Asn Val Ser Ser Trp Thr Val Arg AlaVal Val Lys Val Arg 225 230 235 240 Thr Ile Gly Lys Asp Ile Asn Val LysPro Glu Ile Leu Asp Pro Ile 245 250 255 Thr Asp Thr Leu Asp Val Glu LeuGly Lys Pro Leu Thr Leu Pro Cys 260 265 270 Arg Val Gln Phe Gly Phe GlnArg Leu Ser Lys Pro Val Ile Lys Trp 275 280 285 Tyr Val Lys Glu Ser ThrGln Glu Trp Glu Met Ser Val Phe Glu Glu 290 295 300 Lys Arg Ile Gln SerThr Phe Lys Asn Glu Val Ile Glu Arg Thr Ile 305 310 315 320 Phe Leu ArgGlu Val Thr Gln Arg Asp Leu Ser Arg Lys Phe Val Cys 325 330 335 Phe AlaGln Asn Ser Ile Gly Asn Thr Thr Arg Thr Ile Arg Leu Arg 340 345 350 LysLys Glu Glu Val Val Phe Val Tyr Ile Leu Leu Gly Thr Ala Leu 355 360 365Met Leu Val Gly Val Leu Val Ala Ala Ala Phe Leu Tyr Trp Tyr Trp 370 375380 Ile Glu Val Val Leu Leu Cys Arg Thr Tyr Lys Asn Lys Asp Glu Thr 385390 395 400 Leu Gly Asp Lys Lys Glu Phe Asp Ala Phe Val Ser Tyr Ser AsnTrp 405 410 415 Ser Ser Pro Glu Thr Asp Ala Val Gly Ser Leu Ser Glu GluHis Leu 420 425 430 Ala Leu Asn Leu Phe Pro Glu Val Leu Glu Asp Thr TyrGly Tyr Arg 435 440 445 Leu Cys Leu Leu Asp Arg Asp Val Thr Pro Gly GlyVal Tyr Ala Asp 450 455 460 Asp Ile Val Ser Ile Ile Lys Lys Ser Arg ArgGly Ile Phe Ile Leu 465 470 475 480 Ser Pro Ser Tyr Leu Asn Gly Pro ArgVal Phe Glu Leu Gln Ala Ala 485 490 495 Val Asn Leu Ala Leu Val Asp GlnThr Leu Lys Leu Ile Leu Ile Lys 500 505 510 Phe Cys Ser Phe Gln Glu ProGlu Ser Leu Pro Tyr Leu Val Lys Lys 515 520 525 Ala Leu Arg Val Leu ProThr Val Thr Trp Lys Gly Leu Lys Ser Val 530 535 540 His Ala Ser Ser ArgPhe Trp Thr Gln Ile Arg Tyr His Met Pro Val 545 550 555 560 Lys Asn SerAsn Arg Phe Met Phe Asn Gly Leu Arg Ile Phe Leu Lys 565 570 575 Gly PheSer Pro Glu Lys Asp Leu Val Thr Gln Lys Pro Leu Glu Gly 580 585 590 MetPro Lys Ser Gly Asn Asp His Gly Ala Gln Asn Leu Leu Leu Tyr 595 600 605Ser Asp Gln Lys Arg Cys 610 3 754 DNA Homo sapiens misc_feature (6)..(8)“n” = a, t, c, g 3 tggggnnntg gacagacact gctggcatat ttgtggcaagaagttgatgg ataaaattgt 60 taattaagga ctggactctt ctttattttg aaataaaccatgagtttaag gatggagggg 120 tggactcgga tatgcttgac ttgcacagtt tcttgggtggattctccttt cagttcaatt 180 actcagccag atggcaccac aaagaaatct tgtgggaaaagacagggata ggagctcagg 240 tctgcaaggg gcaggattag tgtgaaagag aatgcacacaagcagatatg gatcaattaa 300 caactaatgt tttaccagca cccacaacct gatgtggcagtcattttagg gggcaagctt 360 tttacatgga aaccggaatt cctaacttac aggtaattagtaaaatgtga agacagaact 420 ccaagacatt tagatcaaag tgtggctgtg cacctaaatcttcatcaagc aggccttcag 480 actttccaat gcaaatagta atctttgttt tcatctttcagtgggagaca ctaaactcaa 540 accagatatt ctggattctg tcnaggacac actgggaagtagaacttgga aagccnttaa 600 ctattanctg caaagcacga tttggctttt aaagggtccttaaccctgtc ataaaatggt 660 acatcaaaga ttctgaccta aaatgggaaa tctcaatacctgaaggcnaa aaagttttaa 720 aatcccacnt ttaaaggatt aaaatcattg ancc 754 4523 DNA Homo sapiens 4 gtaaaatact caagcttttt acatggaaac cggaattcctaacttacagg taattagtaa 60 aatgtgaaga cagagctcca agacatttag atcaaagtgtggctgtgcac ctaaatcttc 120 atcaagcagg ccttcagact ttccaatgca aatagtaatctttgttttca tctttcagtg 180 ggagacacta aactcaaacc agatattctg gatcctgtcgaggacacact ggaagtagaa 240 cttggtaagc tgggcctcat cgcctttgaa tgacatcgtgctgctgggag caggtctaag 300 tgtgatagaa ggaaaacagc attgggattt ccagtcaaacagaattgggt gtgaatctta 360 actcagccat tgactagttt tgtgactttg cacagttacttcatccttta agcctcagta 420 cttagatccg caaatagcta tcataaaact gagcctaaaagattatattg caggccgggc 480 atggtggcct aagcctgtaa tcccagcact ttgggaggctgag 523 5 22 PRT Homo sapiens 5 Val Gly Asp Thr Lys Leu Lys Pro Asp IleLeu Asp Pro Val Glu Asp 1 5 10 15 Thr Leu Glu Val Glu Leu 20 6 2681 DNAHomo sapiens CDS (484)..(2283) 6 ctctctggat aggaagaaat atagtagaaccctttgaaaa tggatatttt cacatatttt 60 cgttcagata caaaagctgg cagttactgaaataaggact tgaagttcct tcctcttttt 120 ttatgtctta agagcaggaa ataaagagacagctgaaggt gtagccttga ccaactgaaa 180 gggaaatctt catcctctga aaaaacatatgtgattctca aaaaacgcat ctggaaaatt 240 gataaagaag cgattctgta gattctcccagcgctgttgg gctctcaatt ccttctgtga 300 aggacaacat atggtgatgg ggaaatcagaagctttgaga ccctctacac ctggatatga 360 atcccccttc taatacttac cagaaatgaaggggatactc agggcagagt tctgaatctc 420 aaaacactct actctggcaa aggaatgaagttattggagt gatgacagga acacgggaga 480 aca atg ctc tgt ttg ggc tgg ata tttctt tgg ctt gtt gca gga gag 528 Met Leu Cys Leu Gly Trp Ile Phe Leu TrpLeu Val Ala Gly Glu 1 5 10 15 cga att aaa gga ttt aat att tca ggt tgttcc aca aaa aaa ctc ctt 576 Arg Ile Lys Gly Phe Asn Ile Ser Gly Cys SerThr Lys Lys Leu Leu 20 25 30 tgg aca tat tct aca agg agt gaa gag gaa tttgtc tta ttt tgt gat 624 Trp Thr Tyr Ser Thr Arg Ser Glu Glu Glu Phe ValLeu Phe Cys Asp 35 40 45 tta cca gag cca cag aaa tca cat ttc tgc cac agaaat cga ctc tca 672 Leu Pro Glu Pro Gln Lys Ser His Phe Cys His Arg AsnArg Leu Ser 50 55 60 cca aaa caa gtc cct gag cac ctg ccc ttc atg ggt agtaac gac cta 720 Pro Lys Gln Val Pro Glu His Leu Pro Phe Met Gly Ser AsnAsp Leu 65 70 75 tct gat gtc caa tgg tac caa caa cct tcg aat gga gat ccatta gag 768 Ser Asp Val Gln Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro LeuGlu 80 85 90 95 gac att agg aaa agc tat cct cac atc att cag gac aaa tgtacc ctt 816 Asp Ile Arg Lys Ser Tyr Pro His Ile Ile Gln Asp Lys Cys ThrLeu 100 105 110 cac ttt ttg acc cca ggg gtg aat aat tct ggg tca tat atttgt aga 864 His Phe Leu Thr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile CysArg 115 120 125 ccc aag atg att aag agc ccc tat gat gta gcc tgt tgt gtcaag atg 912 Pro Lys Met Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val LysMet 130 135 140 att tta gaa gtt aag ccc cag aca aat gca tcc tgt gag tattcc gca 960 Ile Leu Glu Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr SerAla 145 150 155 tca cat aag caa gac cta ctt ctt ggg agc act ggc tct atttct tgc 1008 Ser His Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile SerCys 160 165 170 175 ccc agt ctc agc tgc caa agt gat gca caa agt cca gcggta acc tgg 1056 Pro Ser Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala ValThr Trp 180 185 190 tac aag aat gga aaa ctc ctc tct gtg gaa agg agc aaccga atc gta 1104 Tyr Lys Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn ArgIle Val 195 200 205 gtg gat gaa gtt tat gac tat cac cag ggc aca tat gtatgt gat tac 1152 Val Asp Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val CysAsp Tyr 210 215 220 act cag tcg gat act gtg agt tcg tgg aca gtc aga gctgtt gtt caa 1200 Thr Gln Ser Asp Thr Val Ser Ser Trp Thr Val Arg Ala ValVal Gln 225 230 235 gtg aga acc att gtg gga gac act aaa ctc aaa cca gatatt ctg gat 1248 Val Arg Thr Ile Val Gly Asp Thr Lys Leu Lys Pro Asp IleLeu Asp 240 245 250 255 cct gtc gag gac aca ctg gaa gta gaa ctt gga aagcct tta act att 1296 Pro Val Glu Asp Thr Leu Glu Val Glu Leu Gly Lys ProLeu Thr Ile 260 265 270 agc tgc aaa gca cga ttt ggc ttt gaa agg gtc tttaac cct gtc ata 1344 Ser Cys Lys Ala Arg Phe Gly Phe Glu Arg Val Phe AsnPro Val Ile 275 280 285 aaa tgg tac atc aaa gat tct gac cta gag tgg gaagtc tca gta cct 1392 Lys Trp Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu ValSer Val Pro 290 295 300 gag gcg aaa agt att aaa tcc act tta aag gat gaaatc att gag cgt 1440 Glu Ala Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu IleIle Glu Arg 305 310 315 aat atc atc ttg gaa aaa gtc act cag cgt gat cttcgc agg aag ttt 1488 Asn Ile Ile Leu Glu Lys Val Thr Gln Arg Asp Leu ArgArg Lys Phe 320 325 330 335 gtt tgc ttt gtc cag aac tcc att gga aac acaacc cag tcc gtc caa 1536 Val Cys Phe Val Gln Asn Ser Ile Gly Asn Thr ThrGln Ser Val Gln 340 345 350 ctg aaa gaa aag aga gga gtg gtg ctc ctg tacatc ctg ctt ggc acc 1584 Leu Lys Glu Lys Arg Gly Val Val Leu Leu Tyr IleLeu Leu Gly Thr 355 360 365 atc ggg acc ctg gtg gcc gtg ctg gcg gcg agtgcc ctc ctc tac agg 1632 Ile Gly Thr Leu Val Ala Val Leu Ala Ala Ser AlaLeu Leu Tyr Arg 370 375 380 cac tgg att gaa ata gtg ctg ctg tac cgg acctac cag agc aag gat 1680 His Trp Ile Glu Ile Val Leu Leu Tyr Arg Thr TyrGln Ser Lys Asp 385 390 395 cag acg ctt ggg gat aaa aag gat ttt gat gctttc gta tcc tat gca 1728 Gln Thr Leu Gly Asp Lys Lys Asp Phe Asp Ala PheVal Ser Tyr Ala 400 405 410 415 aaa tgg agc tct ttt cca agt gag gcc acttca tct ctg agt gaa gaa 1776 Lys Trp Ser Ser Phe Pro Ser Glu Ala Thr SerSer Leu Ser Glu Glu 420 425 430 cac ttg gcc ctg agc cta ttt cct gat gtttta gaa aac aaa tat gga 1824 His Leu Ala Leu Ser Leu Phe Pro Asp Val LeuGlu Asn Lys Tyr Gly 435 440 445 tat agc ctg tgt ttg ctt gaa aga gat gtggct cca gga gga gtg tat 1872 Tyr Ser Leu Cys Leu Leu Glu Arg Asp Val AlaPro Gly Gly Val Tyr 450 455 460 gca gaa gac att gtg agc att att aag agaagc aga aga gga ata ttt 1920 Ala Glu Asp Ile Val Ser Ile Ile Lys Arg SerArg Arg Gly Ile Phe 465 470 475 atc ttg agc ccc aac tat gtc aat gga cccagt atc ttt gaa cta caa 1968 Ile Leu Ser Pro Asn Tyr Val Asn Gly Pro SerIle Phe Glu Leu Gln 480 485 490 495 gca gca gtg aat ctt gcc ttg gat gatcaa aca ctg aaa ctc att tta 2016 Ala Ala Val Asn Leu Ala Leu Asp Asp GlnThr Leu Lys Leu Ile Leu 500 505 510 att aag ttc tgt tac ttc caa gag ccagag tct cta cct cat ctc gtg 2064 Ile Lys Phe Cys Tyr Phe Gln Glu Pro GluSer Leu Pro His Leu Val 515 520 525 aaa aaa gct ctc agg gtt ttg ccc acagtt act tgg aga ggc tta aaa 2112 Lys Lys Ala Leu Arg Val Leu Pro Thr ValThr Trp Arg Gly Leu Lys 530 535 540 tca gtt cct ccc aat tct agg ttc tgggcc aaa atg cgc tac cac atg 2160 Ser Val Pro Pro Asn Ser Arg Phe Trp AlaLys Met Arg Tyr His Met 545 550 555 cct gtg aaa aac tct cag gga ttc acgtgg aac cag ctc aga att acc 2208 Pro Val Lys Asn Ser Gln Gly Phe Thr TrpAsn Gln Leu Arg Ile Thr 560 565 570 575 tct agg att ttt cag tgg aaa ggactc agt aga aca gaa acc act ggg 2256 Ser Arg Ile Phe Gln Trp Lys Gly LeuSer Arg Thr Glu Thr Thr Gly 580 585 590 agg agc tcc cag cct aag gaa tggtga aatgagccct ggagccccct 2303 Arg Ser Ser Gln Pro Lys Glu Trp 595ccagtccagt ccctgggata gagatgttgc tggacagaac tcacagctct gtgtgtgtgt 2363gttcaggctg ataggaaatt caaagagtct cctgccagca ccaagcaagc ttgatggaca 2423atggaatggg attgagactg tggtttagag cctttgattt cctggactgg acagacggcg 2483agtgaattct ctagaccttg ggtactttca gtacacaaca cccctaagat ttcccagtgg 2543tccgagcaga atcagaaaat acagctactt ctgccttatg gctagggaac tgtcatgtct 2603accatgtatt gtacatatga ctttatgtat acttgcaatc aaataaatat tattttatta 2663gaaaaaaaac cggaattc 2681 7 599 PRT Homo sapiens 7 Met Leu Cys Leu GlyTrp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg 1 5 10 15 Ile Lys Gly PheAsn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp 20 25 30 Thr Tyr Ser ThrArg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu 35 40 45 Pro Glu Pro GlnLys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro 50 55 60 Lys Gln Val ProGlu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser 65 70 75 80 Asp Val GlnTrp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp 85 90 95 Ile Arg LysSer Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His 100 105 110 Phe LeuThr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro 115 120 125 LysMet Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile 130 135 140Leu Glu Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser 145 150155 160 His Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro165 170 175 Ser Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr TrpTyr 180 185 190 Lys Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg IleVal Val 195 200 205 Asp Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val CysAsp Tyr Thr 210 215 220 Gln Ser Asp Thr Val Ser Ser Trp Thr Val Arg AlaVal Val Gln Val 225 230 235 240 Arg Thr Ile Val Gly Asp Thr Lys Leu LysPro Asp Ile Leu Asp Pro 245 250 255 Val Glu Asp Thr Leu Glu Val Glu LeuGly Lys Pro Leu Thr Ile Ser 260 265 270 Cys Lys Ala Arg Phe Gly Phe GluArg Val Phe Asn Pro Val Ile Lys 275 280 285 Trp Tyr Ile Lys Asp Ser AspLeu Glu Trp Glu Val Ser Val Pro Glu 290 295 300 Ala Lys Ser Ile Lys SerThr Leu Lys Asp Glu Ile Ile Glu Arg Asn 305 310 315 320 Ile Ile Leu GluLys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val 325 330 335 Cys Phe ValGln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu 340 345 350 Lys GluLys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile 355 360 365 GlyThr Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His 370 375 380Trp Ile Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln 385 390395 400 Thr Leu Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys405 410 415 Trp Ser Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu GluHis 420 425 430 Leu Ala Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys TyrGly Tyr 435 440 445 Ser Leu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly GlyVal Tyr Ala 450 455 460 Glu Asp Ile Val Ser Ile Ile Lys Arg Ser Arg ArgGly Ile Phe Ile 465 470 475 480 Leu Ser Pro Asn Tyr Val Asn Gly Pro SerIle Phe Glu Leu Gln Ala 485 490 495 Ala Val Asn Leu Ala Leu Asp Asp GlnThr Leu Lys Leu Ile Leu Ile 500 505 510 Lys Phe Cys Tyr Phe Gln Glu ProGlu Ser Leu Pro His Leu Val Lys 515 520 525 Lys Ala Leu Arg Val Leu ProThr Val Thr Trp Arg Gly Leu Lys Ser 530 535 540 Val Pro Pro Asn Ser ArgPhe Trp Ala Lys Met Arg Tyr His Met Pro 545 550 555 560 Val Lys Asn SerGln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser 565 570 575 Arg Ile PheGln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg 580 585 590 Ser SerGln Pro Lys Glu Trp 595

1-17. Canceled.
 18. An isolated nucleic acid molecule comprising thecoding region of SEQ ID NO:1.
 19. An isolated nucleic acid moleculecomprising the complement of DNA capable of hybridizing under moderatelystringent conditions to the DNA of SEQ ID NO:1 wherein the moderatelystringent condition include 50% formamide and 6×SSC, at 42° C. withwashing conditions of 60° C., 0.5×SSC, 0.1% SDS, and, wherein the DNAencodes a polypeptide that is a cofactor in IL-18 binding.
 20. Anisolated nucleic acid molecule encoding a polypeptide, wherein saidpolypeptide comprises an amino acid sequence that is at least 80%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:2.
 21. An isolated nucleic acid molecule comprising a DNAencoding a polypeptide comprising the amino acids of SEQ ID NO:2.
 22. Anisolated nucleic acid comprising a DNA encoding a polypeptide, whereinsaid polypeptide comprises an amino acid sequence that is at least 80%identical to amino acids x, to 356 of SEQ ID NO:2, wherein x, is aminoacid 1 or
 15. 23. An isolated nucleic acid comprising a DNA encoding asoluble polypeptide comprising amino acids x, to 356 of SEQ ID NO:2,wherein x, is amino acid 1 or
 15. 24. An isolated nucleic acidcomprising a DNA that encodes a polypeptide comprising a fragment of SEQID NO:2, wherein the fragment is a cofactor in IL-18 binding.
 25. Anisolated nucleic acid comprising DNA that encodes a polypeptidecomprising a fragment that is at least 80% identical to SEQ ID NO:2,wherein the fragment is a cofactor in IL-18 binding.
 26. An isolatedpolypeptide comprising an amino acid selected from the group consistingof (a) SEQ ID NO:2. (b) fragments of SEQ ID NO:2, wherein the fragmentis a cofactor in IL-18 binding.
 27. A isolated polypeptide comprising apolypeptide encoded by DNA selected from the group consisting of: (a)the coding region of SEQ ID NO:1; (b) the complement of DNA capable ofhybridizing under moderately stringent conditions to the DNA of SEQ IDNO:1, wherein the moderately stringent condition include 50% formamideand 6×SSC, at 42° C. with washing conditions of 60° C., 0.5×SSC, 0.1%SDS, and, wherein the DNA encodes a polypeptide that is a cofactor inIL-18 binding; and (c) DNA that is degenerate to the DNA of SEQ ID NO:1.28. An isolated polypeptide comprising an amino acid sequence that is atleast 80% identical to a sequence selected from the group consisting of:(a) amino acids x₁ to 356 of SEQ ID NO:2, wherein x₁ is amino acid 1 or15; and (b) fragments of SEQ ID NO:2 wherein the fragment is a cofactorin IL-18 binding.
 29. A polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) amino acids x₁ to 356 of SEQID NO:2, wherein x₁ is amino acid 1 or 15; and (b) fragments of SEQ IDNO:2 wherein the fragment is a cofactor in IL-18 binding.
 30. A fusionprotein comprising the Fc region of Ig a polypeptide selected from thegroup consisting of: (a) amino acids x1 to 356 of SEQ ID NO:2, whereinx1 is amino acid 1 or 15; and (b) a fragment of (a), wherein thefragment is a cofactor in IL-18 binding. (c A polypeptide that is atleast 80% identical to (a), wherein the polypeptide is a cofactor inIL-18 binding.
 31. A recombinant expression vector comprising a DNAselected from the group consisting of: (a) the coding region of SEQ IDNO:1; (b) a DNA encoding amino acids x1 to 356 of SEQ ID NO:2, whereinx1 is amino acid 1 or 15; and (c a DNA encoding an polypeptide that isat least 80% identical to the polypeptide of (b).
 32. A process forpreparing a polypeptide, the process comprising culturing a host celltransformed with an expression vector of claim 31 under conditions thatpromote expression of the polypeptide.
 33. A host cell transformed ortransfected with an expression vector according to claim
 32. 34. Anantibody that is immunoreactive with a polypeptide of claim 26.